Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.
The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.
Inflammation is a beneficial biological response against infectious agents, with the major aim to eliminate the pathogen and repair damaged tissue. Under certain conditions, such as chronic infections or autoimmune diseases, inflammation does not resolve. Instead, there is an aberrant reaction with continuous infiltration of leukocytes, resulting in a prolonged immune response that leads to tissue damage, fibrosis, loss of function, and overall, disability and in some cases death of the patient. These human disorders, cataloged as inflammatory diseases, all involve the blood vessels for the control of leukocyte extravasation1,2.
The endothelial cells play a fundamental role in the regulation of the inflammatory response by controlling leukocyte trafficking. When the endothelial layer is exposed to inflammatory mediators such as LPS, the resting endothelium activates and expresses pro-inflammatory cytokines (Cxcl10, Cxcl5, Cxcl1, etc.) and adhesion molecules (E-selectin, VCAM-1 and ICAM-1) that favor recruitment of circulating leukocytes to the infection site. The leukocytes primed by the released cytokines then mediate rolling and interaction with the endothelial layer through the correspondent adhesive counterparts: PSGL-1 to selectin, α4β1 integrin to VCAM-1, and αLβ2 integrin to ICAM-1. Finally, the leukocytes migrate across the vasculature towards the focus of inflammation3.
The essential role of the endothelium in regulating the inflammatory response has been demonstrated on mice that were genetically modified to express the LPS receptor, toll-like receptor 4 (TLR4), only on the endothelial cells. These endothelial-TLR4 animals were able to respond to an LPS-mediated inflammation and to detect the infection generated after bacteria inoculation, and consequently achieve infection resolution and survival at similar levels as the wild type mice4,5.
For the endothelium-regulated inflammatory response pathway, it has been postulated that the inhibition at some stages of the leukocyte-endothelium interaction would result in the reduction of trans-endothelial migration and a better prognosis for inflammatory-related diseases. In fact, several strategies targeting the endothelial activation and leukocyte-endothelium interaction have been designed to hinder extravasation of immune cells as a treatment for inflammatory disorders6,7.
In this report, we describe a thorough group of in vitro techniques to fully characterize the endothelial activity in response to the inflammatory stimulus LPS and its role in leukocyte activation and adhesion to the vascular layer. The endothelial cell model used in this manuscript was the mouse lung endothelial cell line (MLEC-04), as described by Hortelano et al.8. The MLEC-04 cell line has been validated in the literature to be an appropriate system to study endothelial activation9,10. Based on research interests, these approaches can be easily extrapolated to any endothelial or leukocyte systems and inflammatory profile. Once the endothelial parameters in the selected conditions are defined, the system can test novel drugs on the proposed experimentation to evaluate the vascular activation. In this inflammatory context, the endothelium cells tested with the compound of interest can be compared to the control conditions of the cells, and any resulting differences may inform the drug's prognostic outcome on development and progression of inflammation. To conclude, we propose a relevant system to characterize new drug targets to the endothelial cells, which can influence the design of novel vascular-specific therapies against inflammatory-related diseases.
1. Endothelial Cell Culture
2. LPS Treatment and Mediators
3. Evaluation of Transcriptional Profile on Activated Endothelium by RT-qPCR
4. Assess Endothelial Activation by Flow Cytometry
5. Evaluate Endothelial Activation by Western Blot
6. Evaluate Endothelial Factor Released from Leukocyte Activation by Adhesion Assays
7. Test Endothelial Activation by Leukocyte-Endothelium Co-adhesion Assay
Evaluation of LPS-induced endothelial cell activation by RT-qPCR
The serum starved MLEC-04 cells were stimulated by 100 ng/mL of LPS for 6 h, and the endothelial gene expression was assessed using RT-qPCR by comparing the expression of activation markers to the resting condition. As shown in Figure 1A, the LPS-incubated MLEC-04 cells induced the mRNA expression of selected adhesion molecules involved in leukocyte recruitment during the inflammatory response (E-selectin, VCAM-1 and ICAM-1). PECAM-1 was used as an internal control because the expression is unmodified under this experimental treatment. Figure 1B represents the endothelial activation by LPS measured by the increased mRNA expression of the cytokines Ccl5, Cxcl10, and Cxcl1. These molecules are involved in the activation of circulating leukocytes, including their trafficking to the endothelial layer and later extravasation to the inflammation site. The cytokine, IL4, was used as an internal control under this experimental treatment.
Figure 1: Evaluation of LPS-induced endothelial cells activation by RT-qPCR. The MLEC-04 cells were stimulated with 100 ng/mL of LPS for 6 h (red bars) compared to the control condition (blue bars), and the gene expression was analyzed by RT-qPCR. Graphs show fold induction of mRNA with respect to the control condition of selected endothelial adhesion molecules (A) and cytokines (B) involved in leukocyte activation and recruitment during the inflammatory response. PECAM-1 and IL4 were used as negative controls under this condition. Results are expressed as mean ± SEM from one experiment, out of three performed, carried out in triplicate. Please click here to view a larger version of this figure.
Protein expression of adhesion molecules on LPS-treated endothelial cells
Inflammatory stimulation was performed on the MLEC-04 cells as detailed in the previous section and the protein expression was investigated by western blot technique. The resting endothelium presents almost undetectable levels of the adhesion molecules VCAM-1 and ICAM-1 (labeled as -). In the presence of LPS (+), the endothelial activated phenotype is evident by the upregulation of the expression of the described markers (Figure 2). The membranes were normalized by β-actin detection, which was used as the loading control to calculate the relative band densities after densitometry analysis.
Figure 2: Protein expression of adhesion molecules on LPS-treated endothelial cells. Representative western blot evaluating VCAM-1 and ICAM-1 protein expression in resting (-) compared to LPS-treated (+) MLEC-04 cells. The β-actin was used as a loading control. The molecular weight of each protein is in brackets. The values represent the semi-quantification of relative band densities. Please click here to view a larger version of this figure.
Endothelial surface markers in LPS-mediated inflammation
The endothelial activation in the inflammatory context was evaluated by flow cytometry after 6 hour-stimulation by LPS (100 ng/mL). In this condition, the detection of adhesive-related molecules involved in leukocyte interaction was evaluated. The left panel of Figure 3A represents basal expression of the specified markers in the resting MLEC-04 cells (red-solid histogram) compared to the isotype negative control (black-dotted histogram). The right panel of Figure 3A shows the results derived from stimulated MLEC-04. The LPS treatment significantly upregulated the expression of the adhesion molecules E-selectin, VCAM-1 and ICAM-1 in the plasma membrane (red-solid histogram) when compared to the resting condition (black-dotted histogram). As shown in the cytometry profiles and cited before, the expression of PECAM-1 is unchanged in this experimental condition. Figure 3B shows the quantification values used as references to evaluate possible mediators of the endothelial inflammatory reaction. %: percentage of positive cells; MFI: mean fluorescence intensity.
Figure 3: Endothelial surface markers in LPS-mediated inflammation. Flow cytometry profiles of the cell adhesion molecules on resting and LPS-stimulated MLEC-04 cells. The left panel shows protein expression on the resting cells (Antigen labeled-red histogram) with respect to the isotype matched control (Ctrl-black histogram). The right panel compares the control cell-surface protein expression (black histograms) to the LPS-treated endothelial cells (red histogram). Please click here to view a larger version of this figure.
Signaling pathways triggered by LPS stimulation in endothelial cells
The mechanisms involved in the vascular compartment activation during inflammation are investigated by evaluating key signaling molecules. The MLEC-04 cells stimulated with LPS at different periods were processed for protein extraction, and the degree of activation was analyzed by western blot technique. Figure 4 shows that the IκB-α repressor of NF-κB signaling is phosphorylated after the 15 min LPS-incubation, and returns to basal levels after 1 hour. On the other hand, the LPS activates ERK 1/2 signaling after 15 min, which establishes an essential pathway involved in endothelial activation during the inflammatory response. The membranes were normalized by β-actin or ERK 1/2 detection, which were used as the loading controls to calculate the relative band densities after densitometry analysis.
Figure 4: Signaling pathways triggered by LPS on endothelial cells. The MLEC-04 cells stimulated with 100 ng/mL of LPS for the times indicated in the figure and the signaling pathways ERK 1/2 (P-ERK 1/2) and NF-kB (through P-IκBα) were evaluated by western blot. The ERK 1/2 total and β-actin were used as loading controls in each condition. The molecular weight for each protein is in brackets. The values represent the semi-quantification of relative band densities. Please click here to view a larger version of this figure.
Regulation of the leukocyte behavior by endothelium stimulated with LPS
The biological relevance of the regulation of endothelial activation on the progression of the inflammatory reaction is determined by functional assays of leukocyte performance. As described in the Protocol section, two different approaches are included for testing leukocyte function regulated by endothelial cells. Although the assays interrogate different aspects of the inflammatory response (RT-qPCR for endothelial cytokines released by the leukocyte activation, and western blot for endothelial adhesion molecules in the leukocyte interaction), the data obtained are both evaluate microscopic details of leukocyte attachment. The leukocyte activation by endothelial soluble factors is essential for developing an efficient inflammatory response, as it favors cell adhesion to several substrates. Figure 5A shows the J774 cell adhesion to 0.5 µg/mL fibronectin coated wells in response to previously collected conditioned media from the control or LPS treated endothelial cells. The bright field images and spectrophotometric measurements indicate that the factors released in the conditioned media from LPS-treated endothelium are sufficient to efficiently induce J774 activation, as shown by the induction of cell attachment to fibronectin. Figure 5B represents a co-adhesion assay to evaluate the ability of the vascular layer to support leukocyte firm adhesion by the novel expression of endothelial adhesion molecules. Briefly, the serum starved subconfluent cultures of the MLEC-04 cells stimulated with LPS for 6 h were washed several times and co-incubated with the leukocyte line J774 previously labeled with the fluorescent probe, CFSE. As shown in the micrographs and spectrofluorometric measurements, the LPS-vascular stimulation favors the interaction of CFSE-J774 cells to the endothelial monolayer.
Figure 5: Leukocyte interaction to LPS-incubated endothelial monolayer by co-adhesion assay. (A) Light microscopy images showing crystal violet staining of the J774 cells attached to 0.5 µg/mL fibronectin coated wells in response to conditioned media from the control or LPS-treated endothelial cells. Scale bar, 50 µm. The spectrophotometric analysis shown below indicates the absorbance measurement expressed as arbitrary units (a.u.) ± SEM for a representative experiment run in triplicate. (B) Fluorescence micrographs show attached CFSE-J774 cells to the control or LPS-treated MLEC-04 cells evaluated by co-adhesion assay. Scale bar = 50 µm. The fluorometric analysis shown below indicates the fluorescence intensity expressed as arbitrary units (a.u.) ± SEM for a representative experiment run in triplicate. Please click here to view a larger version of this figure.
Target | NCBI RefSeq ID | Forward sequence (5´-3´) | Reverse sequence (5´-3´) |
GAPDH | NM_001289726.1 | ACT GTG GAT GGC CCC TCT GG3 | TGA CCT TGC CCA CAG CCT TG |
36B4 | NM_007475.5 | AGA TGC AGC AGA TCC GCA T | GTT CTT GCC CAT CAG CAC C |
Cxcl10 | NM_021274.2 | CAA AGC ATC CCG TTT CAC T | CCC CTT CTT GGT GAG GAA TA |
Ccl5 | NM_013653.3 | TCT CTG CAG CTG CCC TCA CC | TCT TGA ACC CAC TTC TTC TC |
Cxcl1 | NM_008176.3 | GGG AAG AAA TGC AAG CTG AA | CTG TAC AGC AGG GTC CTT GAC |
IL1R2 | NM_010555.4 | AGT GCA GCA AGA CTC TGG TAC CTA | AGT TCC ACA GAC ATT TGC TCA CA |
IL10 | NM_010548.2 | CTG GAC AAC ATA CTG CTA ACC G | GGG CAT CAC TTC TAC CAG GTA A |
PECAM-1 | NM_008816.3 | CGA TGC GAT GGT GTA TAA CG | TGT CAC CTC CTT TTT GTC CAG |
E-selectin | NM_011345.2 | GCA TGT GGA ATG ACG AGA GA | GTC AGG GTG TTC CTG TGG TT |
VCAM-1 | NM_011693.3 | GGC TGA ACA CTT TTC CCA GA | CCG ATT TGA GCA ATC GTT TT |
ICAM-1 | NM_010493.3 | CCG CTA CCA TCA CCG TGT A | GGC GGC TCA GTA TCT CCT C |
Table 1: List of primers used in this study.
Resting | LPS | |||
% | MFI | % | MFI | |
Ctrl | 1.79 | 3.5 | 0.55 | 3.48 |
PECAM-1 | 37.52 | 12.09 | 37.82 | 12.24 |
E-selectin | 17.12 | 8.06 | 88.13 | 49.84 |
VCAM-1 | 53.08 | 20.51 | 99.30 | 204.05 |
ICAM-1 | 99.76 | 114.81 | 99.82 | 363.89 |
Table 2: Endothelial surface markers in LPS-mediated inflammation. Quantification of cell adhesion molecules expression on the resting and LPS-stimulated MLEC-04 cells by flow cytometry analysis. The representative values for each marker corresponding to the percentage of positive cells (%) and mean fluorescence intensity (MFI) in the resting and LPS-stimulated endothelial cells. PECAM-1 was used as a negative control under these experimental conditions.
This endothelial protocol describes a stepwise technology that establishes the groundwork for exploring novel mechanisms involved in the regulation of the inflammatory response. These approaches are based on the study of the endothelial activity stimulated by LPS and evaluate the critical steps involved in leukocyte recruitment during the inflammatory response, specifically: endothelial cytokines release, endothelial adhesion molecules expression and leukocyte adhesion to the vascular layer. Once the endothelial parameters are established, the system can search for novel compounds involved in the regulation of endothelial function and consequently, inflammatory progression. Those regulatory drugs can potentially be of interest to the pharmaceutical market. The major projection of this sequential protocol is to establish a foundation for extending these studies to different endothelial types and stimulatory conditions by adjusting to their relative vascular activity parameters.
Drugs selected by this screening will constitute interesting candidates for designing novel therapeutic strategies against inflammatory disorders. On the one hand, the compounds that favor the endothelial response and contribute to leukocyte recruitment will be promising for immunodeficiency conditions16,17. On the other hand, the endothelial inhibitors would constitute excellent therapies for chronic inflammatory diseases10.
Characterization of the cytokines expressed by stimulated endothelium predicts the evolution of the inflammation. Cytokines are small proteins released by the endothelial layer in the inflammatory context and strongly regulate leukocyte behavior. Pro-inflammatory members, such as Ccl5, Cxcl10 and Cxcl1, bind to their specific receptors on the leukocyte surface and signal to induce leukocyte interaction with the newly expressed adhesion molecules on the vascular layer (E-selectin, VCAM-1 and ICAM-1), and leukocyte migration to the pathological area (Figure 1, Figure 2, Figure 3)8,10,18. Other members of the same family of proteins are involved in the resolution of the inflammation and consequently tissue repair. The expression of these anti-inflammatory cytokines is relevant for chronic inflammatory diseases because they impede leukocyte recruitment and favor immunity clearance10,19,20. To select possible endothelial inflammatory regulators, it is recommended to perform this cytokine expression assay and evaluate the endothelial adhesion molecules expression in the presence of the compounds of interest. In fact, analyzing the levels between anti-inflammatory versus pro-inflammatory cytokines can be used as a predictive value for progression of the disease and reveals the drug of therapeutic interest21.
The LPS binding to its cell surface receptor triggers complex intracellular signaling cascades led by MAP kinases ERK 1/2, JNK, and p38 and the NF-kB signalosome to induce the inflammatory transcriptional program. Many of these pathways are common to other inflammatory stimuli such as TNF-α or IL-110,22. The endothelial activation is determined by detecting the phosphorylated form of the MAP kinase members as shown for ERK 1/2 in Figure 4. Moreover, the endothelial activation by LPS induces enzymatic activity of the IKKβ complex, which phosphorylates and degrades the NF-kB inhibitor complex IκB, thus allowing NF-kB effector members of nuclear translocation to perform the correspondent transcriptional activity (Figure 4). Characterizing the mechanisms by which the drug compound affects the endothelial inflammatory response is very useful, not only in describing the drug, but also in designing novel inflammatory treatments in combination with compatible conventional therapies23.
Compounds selected from the endothelial inflammatory screening assays, must be further studied to confirm their functional relevance in the critical steps of leukocyte behavior assays, as ultimately, the cells are responsible for the inflammatory disorders. These assays analyze the leukocyte activity in two different experimental settings. The first is in evaluating the role of endothelial-released cytokines on leukocyte activation by integrin-mediated adhesion assays. The leukocyte integrins are activated by the endothelial released cytokines. Thus, leukocyte adhesive ability to integrin ligands is a representative measurement for leukocyte function8,10,18. The second is in studying the role of the newly expressed endothelial adhesion molecules on the leukocyte interaction to the vascular layer by co-adhesion assays. The endothelial adhesion molecules expressed in the inflammatory context is essential for leukocyte recruitment to the surrounding tissue. Thus, analyzing the leukocytes interacting with the endothelial-treated monolayer of cells constitutes a prognostic value for inflammatory progression (Figure 5)8,10,18. The results derived from these approaches would suggest that the selected compounds are serious candidates in designing future strategies for treating inflammatory disorders
The experimental proposal defined here is a versatile tool for future applications because of its ability to extrapolate to different cellular or stimulatory systems. The procedure can be modified to endothelium from different origins or species, and to test its functionality on several immune cells, as well as different inflammatory agents such as LPS, TNF-α, bacteria, virus, etc. As described in the text, researchers must first characterize the endothelial activation in their own system to later characterize the role of a selected compound on the inflammatory response. Limitations of the protocol are the selection of an appropriate negative control and defining precisely the endothelial activity parameters that discern the resting from the stimulated state. In the case that the conditions described in this paper do not work for a different system, researchers must troubleshoot the experimental parameters by performing additional time-dependent assays and using a concentration gradient of the inflammatory stimulus to define a stimulatory window that allows for testing new drugs for the regulation of the inflammatory response.
To conclude, this endothelial inflammatory protocol is recommended for the search of new endothelial regulators targeting the inflammatory response. Performing this procedure will provide novel, interesting compounds that can be applied to studying its future applications and designing innovative vascular-specific therapies against inflammatory-related diseases, and which potentially could be of interest to the pharmaceutical market.
The authors have nothing to disclose.
This work was supported by the Ministerio de Economía y Competitividad (MINECO) and the Instituto de Salud Carlos III (ISCIII) (grant number IERPY 1149/16 to A.L.; MPY 1410/09 to S. Hortelano); by the MINECO through the Fondo de Investigación en Salud (FIS) (grants numbers PI11.0036 and PI14.0055 to S. Hortelano). S. Herranz was supported by IERPY 1149/16 from ISCIII.
Gelatin | Sigma | G9391 | |
DMEM-F12 | Lonza | BE12-719F | |
Fetal Bovine Serum | Sigma | A4503 | |
Penicillin streptomycin | Lonza | DE17-602E | |
Trypsine | Lonza | BE17-160E | |
EDTA | Sigma | ED2SS | |
LPS | Sigma | L2880 | |
Trizol | Sigma | T9424 | RNA extraction buffer |
Isopropanol | Sigma | 33539 | |
Ethanol absoluto | Panreac | 1,310,861,612 | |
Pure H2O | Qiagen | 1017979 | RNAse free |
Agarose | Pronadisa | 8020 | |
Stain for agarose gels | Invitrogen | s33102 | |
SuperScript III First-Strand Synth | Invitrogen | 18080051 | Reagents for RT-PCR |
Fast SYBR Green Master Mix | Applied Biosystems | 4385610 | Fluorescent stain for qPCR |
MicroAmp Fast Optical 96-Well | Applied Biosystems | 4346906 | Plates for qPCR |
U-bottom 96 well plates | Falcon | 353072 | |
Cytometry tubes | Falcon | 352054 | |
TX100 | Panreac | 212314 | Non-ionic surfactant |
Tris-HCl | Panreac | 1,319,401,211 | |
Sodium chloride | Merck | 1,064,041,000 | |
Sodium pyrophosphate | Sigma | 221368 | |
Sodium fluoride | Sigma | S7920 | |
Sodium orthovanadate | sigma | 13721-39-6 | |
Protease inhibitor cocktail | sigma | P8340 | |
Pierce BCA Protein Assay Kit | Pierce | 23225 | Reagents for bicinchoninic acid assay |
β-mercaptoethanol | merck | 805,740 | |
PVDF Transfer Membrane, 0.45 µm | Thermo Scientific | 88518 | |
Tween-20 | Panreac | 1,623,121,611 | Polysorbate 20 |
PBS | Lonza | BE17-515Q | |
ECL | Millipore | WBKLS0500 | |
Fibronectin | Sigma | F1141 | |
Laminin | Sigma | L2020 | |
Collagen type I | Sigma | c8919 | |
Acetic acid | Panreac | 1,310,081,611 | |
Trypan blue | Sigma | T8154 | |
Paraformaldehyde | Sigma | P6148 | |
Methanol | Panreac | 1,310,911,612 | |
Crystal violet | Sigma | HT90132 | |
Sodium citrate | Sigma | C7254 | |
Ethanol 96% | Panreac | 1,410,851,212 | |
CFSE | Sigma | 21888 | |
RPMI | Lonza | BE12-115F | |
SDS | Bio-Rad | 161-0418 | |
Infinite M200 | Tecan | M200 | Multi mode microplate reader |
Gel Doc 2000 | Bio-Rad | 2000 | Gel documentation system |
StepOnePlus | Applied Biosystems | StepOnePlus | qPCR system |
MACSQuant Analyzer 10 | Miltenyi Biotec | Analyzer 10 | Cytometry equipment |
ChemiDoc MP | Bio-Rad | MP | Chemiluminescence detection system |
Name | Company | Catalog Number | コメント |
Antibodies | |||
PECAM-1 | BD Biosciences | 553370 | Use at 10 µg/ml |
ICAM-2 | Biolegend | 1054602 | Use at 10 µg/ml |
E-selectin | BD Biosciences | 553749 | Use at 10 µg/ml |
VCAM-1 | BD Biosciences | 553330 | Use at 10 µg/ml |
ICAM-1 | Becton Dickinson | 553250 | Use at 10 µg/ml |
anti-rat IgG-FITC | Jackson Immuno Research | 112-095-006 | Use at 10 µg/ml |
anti armenian hamster-FITC | Jackson Immuno Research | 127-095-160 | Use at 10 µg/ml |
Rat IgG isotyope control | Invitrogen | 10700 | Use at 10 µg/ml |
Armenian hamster IgG isotype control | Invitrogen | PA5-33220 | Use at 10 µg/ml |
P-IκΒ-α | Cell Signaling | 2859 | Use at 10 µg/ml |
β-Actin | Sigma | A5441 | Use at 10 µg/ml |
P-ERK | Cell Signaling | 9101 | Use at 10 µg/ml |
anti-mouse HRP | GE Healthcare | LNXA931/AE | Use at 1:10000 |
anti-rabbit HRP | GE Healthcare | LNA934V/AG | Use at 1:10000 |
anti-rat HRP | Santa Cruz | Sc-3823 | Use at 1:10000 |