Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.
Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.
Immunohistochemistry (IHC) is a well-established classic technique used to identify cellular or tissue components (antigens) by antigen-antibody interaction1,2. It can be used to identify the localization and distribution of target biomolecules within a tissue. IHC uses immunological and chemical reactions to detect antigens in tissue sections3. The main markers used for the visualization of antigen-antibody interactions include fluorescent dyes (immunofluorescence) and enzyme-substrate color reactions (immunoperoxidase), both conjugated to antibodies4. Using microscopic observation is possible to determine the localization of labeled tissue, which approximately corresponds to localization of the target antigen in the tissue.
Two methods exist for fluorescent or chromogenic reactions to detect protein: the direct detection method, in which the specific primary antibody is directly labeled; and the indirect detection method, in which the primary antibody is unconjugated while the secondary antibody carries the label5,6,7. The indirect method has some advantages, which is mainly its signal amplification. Moreover, unlike other molecular and cellular techniques, with immunofluorescence, it is possible to visualize the distribution, localization, and coexpression of two or more proteins differentially expressed within cells and tissues7. The choice of the detection method used depends on experimental details.
To date, IHC is widely used in basic research as a powerful and essential tool to understanding the distribution and localization of biomarkers and the general profiling of different proteins in biological tissue from human to invertebrates8,9,10,11. The technique helps display a map of protein expression in a large number of normal and altered animal organs and different tissue types, showing possible down- or up-regulation of expression induced by physiological and pathological changes. IHC is a highly sensitive technique that requires accuracy and the correct choice of methods to obtain optimal results12. First of all, many different factors such as fixation, cross-reactivity, antigen retrieval, and sensitivity of antibodies can lead to false positive and false negative signals13. Selection of the antibodies is one of the most important steps in IHC and depends on the antigen specificity and its affinity to the protein and species under investigation7.
Recently, we have optimized the IHC technique to detect members of orexin/hypocretin and endocannabinoid systems in adult zebrafish tissue. We have focused mainly on fixation, tissue embedding using two different approaches, sectioning and mounting (which can affect resolution and detail during microscopic analysis), and blocking (to prevent false positives and reduce background)14. Other important characteristics are the antibody specificity and selectivity and reproducibility of individual IHC protocols. The key to providing antibody specificity is the use of negative controls (including no primary antibodies or tissue that is known to not express the target proteins) as well as positive controls (including tissue that is known to express the target proteins)15. The selection of antibodies for IHC is made based on their species-specificity (the likelihood with which they react with the antigen of interest) and the antigen-antibody binding detection systems that is used4,5,6,7. In the case of immunoperoxidase, the color of the reaction is determined by selection of the precipitating chromogen, usually diaminobenzidine (brown)16. On the other hand, immunofluorescence utilizes antibodies conjugated with a fluorophor to visualize protein expression in frozen tissue sections and allows for easy analysis of multiple proteins with respect to the chromogenic detection system5,7.
In the immunoperoxidase technique, the secondary antibody is conjugated to biotin, a linker molecule capable of recruiting a chromogenic reporter molecule [avidin-biotin complex (ABC)], leading to amplification of the staining signal. With the ABC reporter method, the enzyme peroxidase reacts with 3,3’-diaminobenzidine (DAB), producing an intensely brown-colored staining where the enzyme binds to the secondary antibody, which can then be analyzed with an ordinary light microscope. ABC staining, due to the high affinity of avidin for biotin, produces a rapid and optimal reaction, with few secondary antibodies attached to the site of the primary antibody reactivity. This chromogenic detection method allows for the densitometric analysis of the signal, providing semi-quantitative data based on the correlation of brown signal levels with protein expression levels18.
With immunofluorescence techniques, simultaneous detection of multiple proteins is possible due to the ability of different fluorochromes to emit light at unique wavelengths, but is important to choose fluorochromes carefully to minimize spectral overlap5. Moreover, the use of primary antibodies in different host species minimizes difficulties concerning cross-reactivity. In this case, each species-specific secondary antibody recognizes only one type of primary antibody. Fluorescent reporters are small organic molecules, including commercial derivatives, such as Alexa Fluor dyes.
Many animal models are used to understand particular physiological and pathological conditions. To date, it is established that many metabolic pathways are conserved over the course of evolution. Therefore, IHC studies in model organisms such as zebrafish can provide insight into the genesis and maintenance of pathological and non-pathological conditions17. It is an aim of this report to illustrate IHC protocols that can be performed on adult zebrafish tissue and used to obtain detailed images of the distribution and localization of OXA, OX-2R, and CB1R at peripheral and central levels. Also reported are protocols for the application of two major IHC indirect methods in peripheral and central tissues of adult zebrafish. Described is the indirect method, which allows for signal amplification in cases where a secondary antibody is conjugated to a fluorescent dye (immunofluorescence method) or enzyme reporter (immunoperoxidase method). Both chromogenic and fluorescent detection methods possess advantages and disadvantages. Reported in this protocol is the use of IHC, mainly immunofluorescence, in adult zebrafish, an animal model widely used to study systems that are evolutionary conserved across different physiological and pathological conditions.
1. Immunoperoxidase protocol
NOTE: The zebrafish were obtained by Prof. Omid Safari (Department of Fisheries, Faculty of Natural Resources and Environment, Ferdowsi University of Mashhad, Mashhad, Iran)10.
2. Immunofluorescence protocol
Representative data for the immunoperoxidase staining are shown in Figure 1 and Figure 2. Immunohistochemical analysis of OX-A and OX-2R distribution in the gut of adult zebrafish showed different localization sites of OX-A and OX-2R and their increases in expression in the intestinal cells of DIO zebrafish. An intense brown staining for OX-A was observed in the cells of the medial and anterior intestine (Figure 1A, A1). The immunoexpression of OX-A gave clear signals in the different gut compartements, decreasing from the anterior toward the medial intestine (Figure 1B, B1). The negative control was used as a reference for the background and to confirm the specificity of OX-A signal (Figure 1E). The prolonged exposure to the chromogen DAB resulted in the increase of the background intensity (Figure 1D). Similar results were observed for OX-2R immunoexpression in the intestine of DIO and control diet zebrafish (Figure 2). An increased OX-A signal in DIO adult zebrafish was accompanied by the overexpression of OX-2R in others intestinal compartements (Figure 2B, B1).
Using immunofluorescence, the data obtained by immunoperoxidase analysis were confirmed, underlying the increase of OX-A and OX-2R expression in the gut of adult DIO zebrafish with respect to the control (Figure 3). Moreover, using double immunofluorescence, it was possible to obtain information about the expression of the endocannabinoid receptor CB1R and its co-localization with OX-A or OX-2R in the gut and brain of control diet and DIO adult zebrafish. The advantage of immunofluorescence is that it yields a more detailed signal with less provided information about tissue morphology, compared to immunoperoxidase technique. Using the immunofluorescence method, we have previously determined anatomical interactions between OX-2R and CB1R in both the gut and brain10.
The accurate analysis of immunofluorescent images showed the increase of OX-2R/CB1R co-localization in the gut of DIO adult zebrafish compared to the control diet zebrafish (Figure 3B, C). A similar situation was observed in different brain regions, such as the dorsal telencephalon, hypothalamus (lateral, ventral, and dorsal zones), optic tectum, torus lateralis, and diffuse nucleus of the inferior lobe (Figure 4). The negative (Figure 5A,B) and positive (mouse brain) (Figure 5C) controls were used as references for the background and to confirm the specificity of CB1R and OX-2R signals.
Moreover, by double immunostaining with OX-A/CB1R, it was observed in the orexinergic neurons of the hypothalamus that there was an increase of co-localization accompanied by an increase of OX-A fluorescent signal (Figure 6). These results show how double immunofluorescence can help to identify physiologically conserved protein expression, co-localization of target proteins, and their distribution and/or expression changes in different pathological conditions.
Figure 1: Orexin immunolocalization in the intestines of DIO vs. control diet adult zebrafish. (A) OX-A immunoreactivity in the cells of the medial intestine of control diet zebrafish. (A1) OX-A immunoreactivity in the cells of the medial intestine of DIO zebrafish. (B) OX-A immunoreactivity in the cells of the anterior intestine of control diet zebrafish. (B1) OX-A immunoreactivity in the cells of the anterior intestine of DIO zebrafish. (A1, B1) An increase of OX-A positive cells in different tissue compartement of the medial and anterior intestine of DIO zebrafish. (C) OX-A immunoreactivity in the cells of the anterior intestine of DIO zebrafish. (D) immunoperoxidase reaction for OX-A after a prolonged exposure to DAB. (E) Negative control. Scale bar: 50 µm (A, A1, B, B1); 100 µm (C, D, E). Please click here to view a larger version of this figure.
Figure 2: Orexin 2 receptor immunolocalization in the intestine of DIO vs. control diet adult zebrafish. (A) OX-2R immunoreactivity in the cells of the medial intestine of control diet zebrafish. (A1) OX-2R immunoreactivity in the cells of the medial intestine of DIO zebrafish. (B) OX-2R immunoreactivity in the cells of the anterior intestine of control diet zebrafish. (B1) OX-2R immunoreactivity in the cells of the anterior intestine of DIO zebrafish. (A1, B1) An increase of OX-2R positive cells in different tissue compartement of the medial and anterior intestine of DIO zebrafish. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 3: Distribution of OX-A (green)/CB1R (red) and OX-2R (red)/CB1R (green) and their co-localization with OX-2R/CB1R (yellow) in the intestines of DIO vs. control diet adult zebrafish. (A) OX-A/CB1R co-expression in the intestine of control diet adult zebrafish. (B) OX-2R/CB1R co-expression within the intestine of control diet zebrafish. (C) OX-2R/CB1R co-expression within the intestine of DIO adult zebrafish. An increase of OX-2R/CB1R co-localization (yellow dots) in the intestine of DIO zebrafish. Scale bar: 25 µm. Please click here to view a larger version of this figure.
Figure 4: Distribution of OX-2R (red) and CB1R (green) and their co-localization with OX-2R/CB1R (yellow) in the brain coronal sections of DIO vs. control diet adult zebrafish. (A) OX-2R/CB1R co-expression in the telencephalon of the control diet zebrafish. (A1) OX-2R/CB1R co-expression in the telencephalon of DIO zebrafish. (B) OX-2R/CB1R co-expression within the lateral, ventral and dorsal zone of hypothalamus, optic tectum, torus lateralis, diffuse nucleus of the inferior lobe of control diet zebrafish (B1) OX-2R/CB1R co-expression within the lateral, ventral, and dorsal zones of the hypothalamus, optic tectum, torus lateralis, and diffuse nucleus of the inferior lobe of DIO zebrafish. (C) Higher magnification of the optic tectum showing the co-expression of OX-2R/CB1R (yellow) in the control diet zebrafish. (C1) Higher magnification of the optic tectum showing the co-expression of OX-2R/CB1R (yellow) in the DIO zebrafish. (D) A particular of the optic tectum showing the distribution and co-expression of OX-2R/CB1R (yellow) in the DIO zebrafish. DAPI (blue) was used to counterstain nuclei. CP: central posterior thalamic nucleus; Dd: dorsal; Dc: central; Dl: lateral; Dm: medial; Dp: posterior part of the dorsal telencephalon; Hd: dorsal zone of the periventricular hypothalamus; Hv: ventral zone of the periventricular hypothalamus; LH: lateral part of the hypothalamus; PGl: lateral and PGm: medial preglomerular nuclei; PGZ: periventricular gray zone of the optic tectum; TeO: optic tectum; TL: torus longitudinalis; Vd: dorsal part of the ventral telencephalon. Scale bar: 50 µm (A, A1, C, C1); 250 µm (B and B1); 25 µm (D). Please click here to view a larger version of this figure.
Figure 5: OX-2R and CB1R protein expression and specificity in adult brain of zebrafish and mouse hippocampus. (A) Negative control of OX-2R by pre-absorption with the relative peptide. (B) Negative control of CB1R by pre-absorption with the relative peptide. (C) Positive control of OX-2R/CB1R in hippocampus of mouse. PGZ: periventricular grey zone of the optic tectum; TeO: optic tectum. Scale bar: 100 µm (A, B); 250 µm (C). Please click here to view a larger version of this figure.
Figure 6: Distribution of OX-A (green) and CB1R (red) and their co-localization with OX-A/CB1R (yellow) in the hypothalamic coronal sections of DIO vs. control diet adult zebrafish. (A) OX-A/CB1R co-expression in the hypothalamus of control diet zebrafish (A1) Higher magnification showing the OX-A/CB1R co-expression within the lateral hypothalamus. Detail of OX-A/CB1R co-expression showing a putative adjacent localization of OX-A/CB1R or co-localization and overlap of OX-A/CB1R in the same cells. (B) OX-A/CB1R co-expression in the Hypothalamus of DIO zebrafish (B1) Higher magnification showing the increased OX-A/CB1R co-expression within the lateral hypothalamus. Detail of OX-A/CB1R co-expression showing a putative adjacent localization of OX-A/CB1R or co-localization and overlap of OX-A/CB1R in the same cells. LH: lateral part of the hypothalamus. Scale bar: 50 µm (A, B); 25 µm (A1, B1). Please click here to view a larger version of this figure.
Sample preparation
Sample preparation is the first critical step in IHC. A reliable protocol allows for maintenance of cell morphology, tissue architecture, and antigenicity. This step requires correct tissue collection, fixation, and sectioning22,23. The purpose of fixation is to preserve tissue and reduce the action of tissue enzymes or microorganisms. In particular, the fixation step preserves cellular components and biomolecules, prevents autolysis and shifting of cell constituents (such as antigens and enzymes), stabilizes cellular materials against aversive effects of the following procedures, and facilitates immunostaining4,7,24.
Before sectioning, the tissue is prepared and preserved through paraffin embedding (immunoperoxidase method) or cryopreserved (freezing in cryomedia, in multiple immunofluorescence methods). The preservation method is associated with the type of fixation7. After fixation and preservation, the tissues are sliced by a microtome if embedded in paraffin, or by a cryostat if embedded in a cryomedia. Tissues are typically sliced at a thickness range of 8-10 μm and mounted on slides. For immunoperoxidase staining, the sample may require additional steps to unmask the epitopes for antibody binding, including deparaffinization and antigen retrieveal25,26. It should be kept in mind that overfixation can cause epitope masking, while underfixation can cause little to no positive signal with heavy edge staining.
Blocking and background reduction
In the immunoperoxidase method, the high affinity of avidin for biotin is likely responsible for the rapid production of background staining. Moreover, since the avidin-biotin reaction is irreversible, the background cannot be removed18,27. During IHC, high background can also be produced by nonspecific binding to endogenous tissue biotines. Hydrophobic and ionic interactions (such as those produced by collagen and other connective tissues such as epithelium and adipocytes), as well as endogenous enzyme activity, are major causes of background staining. Endogenous biotin or enzymes and hydrophobic binding must be minimized prior to antibody staining, which can be achieved by the addition of a detergent, such as Triton X-100, in the blocking buffers28.
The use of 0.3%-0.4% Triton X-100 in the blocking buffers also allows for full permeabilization of the antibodies into tissue sections. Moreover, although antibodies are preferentially specific for one epitope, partial binding to sites on nonspecific proteins is possible, leading to high background staining29. The nonspecific bindings can mask the signal of the target antigen30. To reduce nonspecific background staining, samples should be incubated with a buffer that blocks nonspecific reactive sites. Common blocking buffers include normal serum or bovine serum albumin30. The species of the blocking serum should be the same as the host of the secondary antibody. It is recommended to determine the best incubation time. Concentration of the normal serum in the blocking buffer is another important determination31. Furthermore, to eliminate the background staining, it is crucial to use the optimal dilution of the primary and secondary antibodies. Thus, incubation time must be chosen carefully, in addition to the temperature (i.e., increase the time if performing at 4 °C, decrease the time if at RT) and detection system.
Antibody choice
The selection of antibodies for IHC staining is important and can affect experimental outcome32. To ensure that the antibody will respond appropriately, the epitope recognized by it must be considered. Understanding the target protein and its function, tissue and subcellular localization, and whether it undergoes post-translational modifications can help to determine the choice of antibody. Another important step is the testing of different concentrations of primary/secondary antibody to keep the background and aspecificity at a minimum level compatible with a specific signal. Moreover, it is important to check species reactivity to confirm primary and secondary antibody compatibility and the capability of the primary antibody to recognize the antigen target in its native conformation. Another important step for primary antibody choice is gene alignment. This ensures that the primary antibody reacts with the biomolecule of interest and provides information about which epitope is recognized by the antibody in a specific animal model. Gene alignment also provides the possibility of choosing antibodies capable of recognizing epitopes that are evolutionary conserved.
Controls
To clarify specificity of the antibodies, an important aspect is performance of the controls, which allows detection of specific staining. The controls include: i) a tissue known to express the antigen as a positive control; ii) a tissue known not to express the antigen as a negative control; iii) the omission of the primary antibody or absorption of the primary antibody with a specific peptide to confirm that the secondary antibody does not crossreact with other tissue components14,33.
In IHC techniques, several steps can cause problems prior to achieving the final staining. Strong background staining and nonspecific target antigen staining can be caused by endogenous biotin or primary/secondary antibody cross-reactivity and poor enzyme activity or primary antibody potency, respectively. Background staining and nonspecific binding can be prevented by blocking the endogenous enzymes prior to the incubation with the primary antibody. Using normal serum is the best way to block nonspecific interactions30. The choice of the blocking buffer depends on the method of detection used. Moreover, a tissue known to lack the expression of the target antigen as negative control can act as the reference to determine the amount of background staining34. The deposition of chromogenic or fluorescent signal in the negative control confirms the presence of nonspecific staining. Furthermore, insufficient blocking time blocking leads to a high background, while excessive blocking leads to a low signal35.
In immunoperoxidase staining, the presence of endogenous peroxidase in the tissue is another cause of brown background deposition. The treatment with H2O2 prior the incubation with the primary antibody blocks the endogenous peroxidase36,37. On the contrary, in immunofluorescence, fixation plays a key role in the generation of autofluorescence. To avoid autofluorescence, the best fixation method, time of fixation, and preparation of tissues must be carefully chosen. Another critical step of IHC is validation of the primary antibody. An antibody is considered valid if it produces a consistent and specific staining pattern in a particular tissue or cell/subcellular components and if pre-absorption of the primary antibody with a specific peptide does not yield staining38. In immunofluorescence, nonspecific binding shows similar fluorescent intensity under three color fluorescence detectors, while the signal is variable since different fluorescent conjugated secondary antibodies are used. These aspects of IHC tissue preparation and antibody staining must be addressed to overcome staining issues.
IHC, although a relatively simple technique, presents some limitations and depend on many factors39. One of the crucial points is formalin fixation, which can alter the expression of post-translation modified proteins. On the other hand, formalin-fixed paraffin-embedded tissues can be stored for long-term at room temperature, whereas frozen tissues can only be stored for up to one year at -80 °C. Moreover, frozen tissues can be damaged by the formation of ice crystals, which can affect the subcellular details altering IHC staining40,41. Regarding our studies, the most difficult aspect was finding specific primary antibodies against zebrafish molecules. Although zebrafish have been recognized as a valid animal model with a highly conserved degree of structure, very few antibodies have been developed that can recognize specific proteins and other molecules in zebrafish. To overcome these limitations, it is important to validate antibody specificity by western blotting analysis and gene alignment.
Growing interest in IHC methods has led to the development of highly specific immunostaining that can help investigative studies42. IHC is being used with increasing frequency to identify the presence of specific molecular markers and their changes across different pathologies. The two approaches illustrated here, immunoperoxidase and immunofluorescence, have respective advantages and disadvantages43. Paraffin-embedded tissue, used in the immunoperoxidase technique, can allow for high resolution of cells and tissue and reveal details about the distribution and amount of target proteins44,45. However, paraffin-embedded tissues are not suitable for immunofluorescence, since paraffin can mask the antigenicity and lead to nonspecific fluorescence46. On the other hand, cryosection of PFA-fixed tissue preserves endogenous antigenicity and lead to a decrease in nonspecific fluorescence. Even if the technical quality of the cryosections are much lower than that of paraffin-embedded tissue, they can yield valid results using the IHC technique47.
Moreover, while paraffin embedding better preserves morphological details, cryopreservation better preserves enzyme and antigen expression, leading to more detailed immunostaining. The immunofluorescence technique also allows the contemporary detection of two or three different biomolecules, revealing possible interactions, as illustrated in our previous work for orexin and endocannabinoid systems10. Among the techniques used to detect the distribution and levels of specific biomolecules, IHC allows not only the determination of specific morphological expression and distribution of molecules and proteins, but also the possibility to perform quantitative analysis.
Using immunofluorescence, the codistribution and coexpression of biomolecules can also be further understood, as well as possible interactions and their changes in cases of different pathologies48. Confocal microscopy, during the last 20 years, has often been used to study the cellular and subcellular distribution of numerous proteins in the mammalian brain. Fluorescence microscopy also allows the visualization (using fluorophores or fluorescent dyes) of specific structures of interest, such as proteins, organelles, and other biological matter; such signals can be used in both fixed and living biological systems to image specific subcellular structures49. The potential modulatory function of biomolecules in specific cell compartments can be explored via the visualization of their expression patterns, whereas the role of protein signaling within the tissues may be uncovered via the neuroanatomical distribution of metabolic enzyme expression or receptors.
The presented technical work introduces IHC approaches, mainly immunofluorescence, to studying two highly conserved systems, orexin and endocannabinoid, in an adult zebrafish model. In particular, immunofluorescence methods can be used to determine the distribution, relative amount, and anatomical interactions of specific proteins in target tissues. Cryopreservation of PFA-fixed tissues better preserves highly sensitive proteins susceptible to rapid deterioration. Moreover, cryopreservation is thought to better preserve antigen and antigenicity, and it allows for studying of post-translation modified proteins and DNA.
Even if frozen tissues (compared to paraffin-embedded sections) are thicker, which hampers the ability to observe tissue morphology in detail, confocal microscopy allows for sample visualization in great detail and enhances imaging capabilities. Moreover, immunofluorescence can be used for quantitative analysis of staining intensity, and densitometric analysis of the signal provides quantitative data. This allows for determining correlations of fluorochrom signal levels with protein expression levels by looking at areas of co-localization. This work describes and illustrates protocols that can be used to study evolutionary conservation of important proteins in the adult zebrafish. The use of IHC, mainly immunofluorescence, in adult zebrafish can help highlight the usefulness of this animal model in studying the morphological expression and distribution of highly conserved biomolecules, as well as reveal possible alterations across different pathological conditions correlating with human physiopathology.
The authors have nothing to disclose.
This study was supported by Fondi Ricerca di Ateneo (FRA)2015-2016, University of Sannio.
Anti CB1 | Abcam | ab23703 | |
Anti OX-2R | Santa Cruz | sc-8074 | |
Anti-OXA | Santa Cruz | sc8070 | |
Aquatex | Merck | 1,085,620,050 | |
Biotinylated rabbit anti-goat | Vector Lab | BA-5000 | |
citric acid | Sigma Aldrich | 251275 | |
Confocal microscope | Nikon | Nikon Eclipse Ti2 | |
Cryostat | Leica Biosystem | CM3050S | |
DAPI | Sigma Aldrich | 32670 | |
Digital Camera | Leica Biosystem | DFC320 | |
Digital Camera for confocal microscope | Nikon | DS-Qi2 | |
Donkey anti goat Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A11055 | |
Donkey anti goat Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A11058 | |
Donkey anti rabbit Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A21206 | |
Donkey anti rabbit Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A21207 | |
Ethanol absolute | VWR | 20,821,330 | |
Frozen section compound | Leica Biosystem | FSC 22 Frozen Section Media | |
H2O2 | Sigma Aldrich | 31642 | |
HCl | VWR | 20,252,290 | |
ImmPACT DAB | Vector lab | SK4105 | |
Microscope | Leica Biosystem | DMI6000 | |
Microtome | Leica Biosystem | RM2125RT | |
Na2HPO4 | Sigma Aldrich | S9763 | |
NaCl | Sigma Aldrich | S7653 | |
NaH2PO4H2O | Sigma Aldrich | S9638 | |
NaOH | Sigma Aldrich | S8045 | |
Normal Donkey Serum | Sigma Aldrich | D9663 | |
Normal Rabbit Serum | Vector lab | S-5000 | |
paraffin wax | Carlo Erba | 46793801 | |
Paraphormaldeyde | Sigma Aldrich | P6148 | |
sodium citrate dihydrate | Sigma Aldrich | W302600 | |
Triton X-100 | Fluka Analytical | 93420 | |
Trizma | Sigma Aldrich | T1503 | |
VectaStain Elite ABC Kit | Vector lab | PK6100 | |
Xylene Pure | Carlo Erba | 392603 |