Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

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.

1. Tissue dissection
   1. Sacrifice the zebrafish by submersion in ice water (5 parts ice/1 part water, 4 °C); leave them until cessation of all movement to ensure death by hypoxia.
   2. Quickly remove the gut and brain with the following dissection method:
      1. Dry the fish on a paper towel and place it sagittally on a dissecting mat, blocking the ventral part of the eye socket and fleshy part of the tail.
      2. For the intestine: cut the skin and carefully remove the skin and underlying muscle from the side of the fish until the internal organs are visible. Remove the intestine from the body cavity stretching it out.
      3. For the brain: remove the head with a razor blade. Remove soft tissue from the ventral side of the skull with forceps. Open the skull and remove the bone from the ventral side of the brain. Remove the skin and skull bones from the dorsal side of the brain. Remove the brain.
2. Tissue fixation
   1. Fix the dissecting tissues by immersion in fresh 4% paraformaldeyde (PFA) in phosphate buffer (PB, pH 7.4, 4 °C) for 3 h at room temperature (RT).
      1. Prepare 0.1 M PB by dissolving 1.755 g of NaH₂PO₄H₂O and 4.575 g of Na₂HPO₄ in 450 mL of distilled water (dH₂O), and adjust to a pH of 7.4 with the necessary amount of NaOH 1N.
      2. Prepare 4% PFA dissolving 4 g of PFA in 100 mL of 0.1 M PB (pH 7.4) by agitation on a heat plate. When a temperature of 60 °C is reached, add 2-4 drops of NaOH 1N to obtain a clear solution. Left PFA 4% at RT and control the pH . Adjust the pH to 7.4.
         NOTE: Tissue fixation for more than 4–6 h may lead to overfixation, which masks antigens, limiting antibody-epitope binding. Time of fixation depends on tissue size.
3. Tissue embedding
   1. Rinse the tissues 5x for 5 min each, by immersion in 0.1 M PB (pH 7.4).
   2. Dehydrate the tissues by subsequent immersion in alcohol 70% (6 min), 80% (6 min), 95% (5 min), 95% (5 min), 100% (1 min), and 100% (1 min).
   3. Clarify the tissues by immersion in 100% alcohol:xylene (1:1) for 10 min, then 2x in xylene for 5 min each.
   4. Infiltrate the tissues with paraffin wax (56 °C) twice for 1 h each by direct immersion in paraffin.
   5. Embed the tissues in paraffin blocks at room temperature (RT) and store at RT until sectioning.
4. Tissue cutting
   1. Cut tissues into coronal or sagittal sections with 8 µm thickness with a microtome, and collect the sections in alternate series onto adhesive glass slides on the water and warmed at 38 °C.
   2. Dry the slides with sections at 37 °C overnight.
5. Deparaffinization and rehydratation of tissue sections
   1. Dewax the sections by immersion in xylene for 5 min followed by immersion in xylene for 3 min.
   2. Rehydrate the sections by subsequent slides immersion in alcohol 100% II (1 min), 100% I (1 min), 95% (1 min), 75% (1 min), 50% (1 min), then place the slides in dH₂O (5 min).
      NOTE: Completely dewax the sections before starting the reaction. If the sections still have traces of paraffin, perform an additional immersion in xylene for 5 minutes or more.
6. Antigen retrieval
   1. Treat the sections with citrate buffer (0.01 M, pH 6.0) by immersing the slides in the solution and heating in a microwave oven for 5 min at maximal power.
   2. Let the sections cool.
   3. Repeat the cycle of 5 min in the microwave at maximal power to complete the antigen retrieval.
      1. Prepare citrate buffer (0.01 M, pH 6.0) by dissolving 2.10 g of citric acid in 100 mL of dH₂O and 5.882 g of sodium citrate in 200 mL of dH₂O. Mix sodium citrate (0.01 M) with citric acid (0.01 M) at a 1:4 ratio by volume and adjust the pH to 6 using HCl 0.1N.
7. Blocking endogenous peroxidase
   1. Demarcate the tissue area on the slides with a solvent-resistant pen.
   2. Rinse the sections 3 times, 5 min each, with tris-buffered saline solution (TBS) (0.1 M, pH 7.3).
      1. Prepare 0.1 M TBS by dissolving 12.1 g of trizma base and 9 g of NaCl in 950 mL of dH₂O and adjust to pH 7.3 with HCl 0.1N.
   3. Block the endogenous peroxidase activity by slides immersion in a solution of 0.75% H₂O₂ for 5 min at RT.
      1. Prepare the 0.75% H₂O₂ solution dissolving 5 mL of 30% H₂O₂ in 195 mL of dH₂O.
         NOTE: ndogenous enzymes can react with IHC reagents and yield false positive results. Moreover, highly vascularized tissues express many endogenous peroxidase, which can lead to intense nonspecific staining and background levels. Treatment with 0.75% H₂O₂ quenches endogenous peroxidase and significantly reduces the nonspecific background.
8. Blocking of nonspecific binding sites and tissue permeabilization
   1. Incubate the tissue sections with the TBS/0.4% Triton (TBS-T) blocking buffer, containing the primary antiserum (NRS), for 30 min at RT to block nonspecific binding sites.
      1. Prepare 0.4% Triton by dissolving 0.4 mL of Triton X-100 in 100 mL of TBS.
      2. Prepare the blocking buffer TBS-T solution by dissolving 1% normal serum in TBS containing 0.4% Triton X-100 (1 mL of normal serum + 8.2 mL of TBS + 0.8 mL of 0.48 Triton X-100).
         NOTE: Select the species of the animal serum depending on the host of the secondary antibody (e.g., when using a goat anti-rabbit secondary antibody, use normal goat serum).
9. Tissue incubation with primary antibody
   1. Incubate the sections overnight in a humid box at RT with primary antibodies diluted in TBS-T. Stain the sections with the following primary antibody:
      1. Goat antibody against OX-2R diluted 1:200, which recognizes the C-terminal of the protein.
      2. Goat antibody against OX-A diluted 1:200 [orexin-A (C-19)], which recognizes the C-terminal of the protein.
         NOTE: Ensure that the primary antibody reacts with the biomolecule of interest. Define with which epitope of the biomolecule the primary antibody reacts by using the gene alignment. For instance, the OX-A epitope has been mapped between the aa 50–100 of human OX-A (O43612) whereas the OX-2R epitope has been mapped near the last 50 aa at the C-terminal of human.
10. Tissue incubation with secondary antibody
    1. Rinse the sections 3x for 5 min each, with 0.1 M TBS (pH 7.3).
    2. Incubate the sections for 1.5 h at RT with biotinylated secondary antibody and rabbit anti-goat immunoglobulin (H+L) conjugated with biotin, then dilute 1:100 in TBS-T.
       NOTE: Test and find the different dilutions of both primary and secondary antibodies to find the optimal dilution that allows reduction of the background.
11. Peroxidase reaction
    1. Rinse the sections 3x for 5 min each, with 0.1 M TBS (pH 7.3).
    2. Incubate the sections with avidin-biotin complex (ABC) for 1 h.
       1. Prepare ABC solution adding two drops of component A followed by two drops of component B in 5 mL of TBS.
    3. Rinse the sections 3x for 5 min each, with 0.1 M TBS (pH 7.3).
    4. Treat the sections with the chromogen substrate 3,3’-diaminobenzidine-4 (DAB).
       1. Prepare DAB solution adding one drop (approximately 30 µL) of DAB chromogen concentrate to 1 mL of DAB diluent, and mix well before use.
          NOTE: Prepare ABC solution at least 30 min before use and keep the complex at 4 °C until use to allow for stable avidin/biotin binding. Prepare the fresh DAB working solution, apply to the tissue sections, and develop until the color is appropriate. When the chromogenic reaction converts the epitope sites to a brown color and the intensity of the signal is appropriate for imaging, proceed to the next step. Timing of DAB development may vary from a few seconds to 10 min. For OX-A and OX-2R 1, 2 min is enough. Handle DAB with care, as it is carcinogenic.
12. Tissue dehydratation and mounting
    1. Rinse all the sections 3x for 5 min each, with 0.1 M TBS (pH 7.3) to stop the DAB reaction.
    2. Dehydrate the sections by subsequent slides immersion in alcohol 50% (2 min), 75% (2 min), 95% (2 min), 100% I (2 min), 100% II (2 min).
    3. Clarify the slices by immersion 2x in xylene 10 min each.
    4. Mount the sections with DPX (dibutyl phthalate xylene) mountant for histology, adding two drops of mounting media to slides and topping slowly with coverslips.
       NOTE: Since the immersion of tissue in increasing concentrations of alcohol during dehydration results in alcohol penetration of tissue and replacement of water with alcohol, determine a precise dehydration time and do not over dehydrate the tissues. Perform the immersion in xylene after dehydration to clarify the tissue and remove any excess or remaining alcohol.
13. Controls
    1. Repeat sections 1.1–1.12, omitting the primary antibody and/or substituting the primary antibody or the secondary antibody IgG by TBS (negative controls).
    2. Repeat sections 1.1–1.12 pre-absorbing each primary antibody with an excess of the relative peptide (100 mg of peptide/1 mL of diluted antiserum).
    3. Repeat sections 1.1–1.12 using different tissue as positive control (e.g., mouse tissue).
       NOTE: Prepare all buffers fresh, shortly before starting. Carefully remove the excess fluid at each step with a pipette or filter paper around the sections, always keeping the sections wet.
14. Image acquisition and analysis
    1. Acquire digital images under constant light illumination and at the same magnification using a microscope equipped with a digital camera.
    2. Perform a semi-quantitative analysis of staining intensity using imaging software (see Table of Materials).
       1. Open the captured images, in .lif or .tiff file format, for evaluating indices of OX-A or OX2-R positivity.
       2. Select the button under Measure and click on Manual Counting. Count the number of stained cell profiles directly from the screen by placing a mark onto positive cells by clicking the mouse.
       3. Select a region of interest (ROI) area (3 x 10³ μm²) to quantify the immunosignal density (optical density, OD).
       4. Determine the pixel with the highest intensity (high positive) and least intensity (negative) inside the analyzed image to assign the zero as the value of the background (i.e., a portion of tissue devoid of stained cells).
       5. Assess the OD by working on a logarithmic scale of absorbance. In digital image analysis, the DAB pixel intensity ranges from the darkest (0 value) to lightest (255 value) shade.
       6. Determine OD by plotting a histogram of the following: log₁₀(255/I), where “I” is the pixel intensity value given by the program and determined by subtracting the background.
          NOTE: The permanent and stable immunoperoxidase reaction can be analyzed under a bright-field microscope at any time. Perform the counting of labeled cells on alternate section to avoid double-counting of the same cell from adjacent sections.

2. Immunofluorescence protocol

1. Tissue dissection
   1. Sacrifice and dissect the animals as described in section 1.1.
2. Tissue fixation
   1. Fix the gut and brain by immersion for 3 h in 4% PFA at 4 °C.
      1. Prepare PB and 4% PFA as in section 1.2.1.
         NOTE: The time of fixation depends on tissue size. Tissue fixation for more than 4–6 h may lead to overfixation, which masks antigens and limits antibody-epitope binding.
3. Tissue embedding
   1. Rinse the tissues 3x for 5 min each, with 0.1 M PB (pH 7.4).
   2. For the cryoprotection, transfer the tissues to 20% sucrose in PB (0.1 M, pH 7.4) and keep overnight at 4 °C. Then, transfer the tissues to 30% sucrose in 0.1 M PB (pH 7.4) and keep for an additional night at 4 °C.
   3. Embed the tissues in a block of optimal cutting temperature (OCT) compound. To do this, prepare a small dewar of liquid nitrogen, take aluminum foil, and cut it in half to create a pan. The pan containing the tissues filled with the OCT compound has to be dipped in the liquid nitrogen until the OCT compound is cooled. The frozen tissues can be stored at -80 °C until sectioning.
4. Tissue cutting
   1. Transfer the frozen tissue blocks to a cryostat at -20 °C and cut in coronal or sagittal sections of 10 μm.
   2. Collect the tissues in alternate serial sections onto adhesive glass slides suitable for immunohistochemistry and store them at -20 °C until use.
      NOTE: Store slides between -20 °C and 4 °C in a dark slide box or slide book.
5. Blocking of nonspecific binding sites and tissue permeabilizzation
   1. Demarcate the tissue area on the slide with a solvent resistant pen.
   2. Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
   3. Incubate the sections with 1% normal donkey serum dissolved in the permeabilization buffer PB-Triton X-100 0.3% (PB-T) for 30 min at RT to permeabilize the cell membrane and block the nonspecific binding sites.
      1. Prepare Triton X-100 0.3% dissolving 0.3 mL of Triton X-100 in 100 mL of 0.1 M PB (pH 7.4).
      2. Prepare the blocking solution dissolving 1% normal donkey serum in PB containing 0.3% TritonX-100.
         NOTE: The animal species of the serum used in the permeabilization and blocking buffers are dependent on the host of the secondary antibody.
6. Incubation with mix of primary antibodies
   1. Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
      1. Incubate the sections overnight in a humid box at RT with a mix of primary antibodies diluted in PB-T. The following mixes of primary antibodies can be used: goat antibody against OX-2R diluted 1:100/rabbit antibody against CB1R diluted 1:100, or goat antibody against OX-A diluted 1:100/rabbit antibody against CB1R diluted 1:100.
7. Incubation with mix of secondary antibodies
   1. Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
   2. Incubate the sections for 2 h at RT with 1) a mix of donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody and donkey anti-goat Alexa Fluor 594-conjugated secondary antibody diluted 1:100 in PB-T; or 2) a mix of donkey anti-goat Alexa Fluor 488-conjugated secondary antibody and donkey anti-rabbit Alexa Fluor 594-conjugated secondary antibody diluted 1:100 in PB-T.
      NOTE: Use secondary antibodies developed in the same animal host. The normal serum must belong to the same species of the secondary antibody (e.g., use secondary antibodies developed in donkey and a donkey normal serum). Dilute the primary and secondary antibodies with blocking buffer containing the detergent to increase cell permeabilization and reduce background.
8. Tissue mounting
   1. Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
   2. Counterstain the sections with nuclear dye DAPI (4’,6-diamidino-2-phenylindole) prepared dissolving 1.5 µL of DAPI (1 mg/mL) in 3 mL of PB.
   3. Coverslip slides with mounting medium (see Table of Materials). This aqueous mounting medium stabilizes the tissue sample and stains for long-term usage. Fluorescent samples can be stored in the dark at 4 °C. To prolong the life of fluorofores, use an antifed mounting medium.
      NOTE: Choose a good mounting medium. One of the most important parameters of mounting agents is the refractive index (nD), which should be around 1.5, the refractive index of glass. The mounting medium used here can be used especially with specimen prepared for enzyme and lipid determinations (i.e., specimen that must not be dehydrated with an ascending series of alcohol).
9. Controls
   1. Repeat sections 2.1–2.8, omitting the primary or secondary antibody, or substituting in the specific step the primary or secondary antisera with PB (negative control).
   2. Repeat sections 2.1–2.8, pre-absorbing each primary antibody with an excess of the relative peptide (100 mg of peptide/1 mL of diluted antiserum).
   3. Repeat sections 2.1–2.8 on slices of mouse brain (positive control).
      NOTE: Prepare all the buffers fresh, shortly before starting. Remove the excess of fluid carefully at each step with a pipette or filter paper around the sections, always keeping the section humid. 
10. Image acquisition
    1. Use a confocal microscope equipped with an x-y-z motorized stage, digital camera, and acquisition and image analysis software such as NIS-Elements C to observe and analyze the immunostained sections. Acquire digital images using the 5-20-40x objectives.
    2. Take the images of each section at low magnification (10x or 20x objective) in each of the available channels to compose a low magnification montage, providing an overview of the entire region to facilitate the localization and documentation of OX-2R/CB1R anatomical co-expression. Normalize the fluorescence images to maximum contrast and overlay before the analysis.
    3. Collect serial Z-stacks of images throughout the area of interest. Acquire the images through six focal planes (Z-step) with focal steps of 1–1.8 µm to cover the tissue volume in which OX-2R/CB1R coexpression is visualized as yellow puncta. To do this collection for each channel (red, green, blue) separately, by using a Z-motorized microscope.
    4. Use an imaging deconvolution software to deconvolve images by application of ten iterations and collapse the serial Z planes images into a single maximum projection image.
    5. Adjust the micrographs for light and contrast using Adobe Photoshop 6.01 (Adobe Systems, San Jose, CA).
       NOTE: Perform the deconvolution step during observation to further decrease the background. Limit the slide exposure to light to prevent photobleaching.
11. Image analysis
    1. Perform quantitative analysis of OX-2R/CB1R coexpression on alternated 10 μm thick sections (n = 5 animal per group) by covering all the region of interest of each animal.
    2. Quantify the OX-2R/CB1R coexpression as number of yellow positive puncta with a semiautomated system of image analysis. Imagins software can provide a higher level of detail, with quantitative data regarding the regions of overlap between different fluorescent probes.
    3. Quantify the puncta by using thresholding tools for signal intensity in the two channels. Open the .liff, .tiff, or .jpg images file and select Image–Threshold. Select Auto Setting or Manual Method and regulate Threshold until all the stained puncta are selected. Analyze the distribution of the pixel intensities in an area of the image that does not contain any immunolabelled objects to obtain the background threshold. Determine this background individually for every image. The program then removes the background threshold by setting the baseline of pixel intensities to the background value.
    4. Select Analyze–Measure and choose the parameters to be measured (puncta intensity-minimum, maximum and mean values; number/density of the immunolabelled puncta).
    5. Count the yellow puncta along the volume of the tissue in 4 Z-stacks for each section, using the stacks immediately above or below to the best focus plane. Exclude the out-of-focus regions from the analysis.
       NOTE: Perform the counting of labeled cells on alternate section to avoid double-counting of the same cells from adjacents sections.