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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence protocol
- Tissue dissection
- 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.
- 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.
- Tissue fixation
- Fix brain by immersion for 3 h in 4% paraformaldehyde (PFA) at 4 °C.
- Prepare 0.1 M phosphate buffer (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.
- 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 room temperature (RT) and control the pH . Adjust the pH to 7.4.
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.
- Tissue embedding
- Rinse the tissues 3x for 5 min each, with 0.1 M PB (pH 7.4).
- 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.
- 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.
- Tissue cutting
- Transfer the frozen tissue blocks to a cryostat at -20 °C and cut in coronal or sagittal sections of 10 μm.
- 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.
- Blocking of nonspecific binding sites and tissue permeabilization
- Demarcate the tissue area on the slide with a solvent resistant pen.
- Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
- 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.
- 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).
- 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.
- Incubation with mix of primary antibodies
- Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
- 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 orexin receptor (OX-2R) diluted 1:100/rabbit antibody against cannabinoid receptor (CB1R) diluted 1:100, or goat antibody against orexin peptide (OX-A) diluted 1:100/rabbit antibody against CB1R diluted 1:100.
- Incubation with mix of secondary antibodies
- Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
- 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 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.
- Tissue mounting
- Rinse the sections 3x for 5 min each, with 0.1 M PB (pH 7.4).
- 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.
- 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 fluorophores, use an antifade 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).
- Controls
- Repeat sections 1.1–1.8, omitting the primary or secondary antibody, or substituting in the specific step the primary or secondary antisera with PB (negative control).
- Repeat sections 1.1–1.8, pre-absorbing each primary antibody with an excess of the relative peptide (100 mg of peptide/1 mL of diluted antiserum).
- Repeat sections 1.1–1.8 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.