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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
- Tissue Staining
- In a light-tight staining box, create a “humidity chamber” with lint-free tissues soaked with deionized water.
- Dry the edges of the slide with a lint-free tissue, use a mini pap pen to make a liquid-repellent border at the very edge of the slide, away from tissue sections. This border should ensure ample space between the meniscus of the liquid and the edge of the tissue so that surface tension does not affect staining.
NOTE: The pap pen-repellent border can be applied prior to 1.1.3 if the antibodies of interest do not require microwave antigen retrieval. If the pap pen has been applied prior to washing in PBS, the integrity of the liquid-repellent border must be checked at this step. Use a mini-pap pen to fill in any gaps in the border. - With slides laid horizontally, block nonspecific antigen binding by incubating in 4% v/v serum in PBS (block solution). Pipette 300 µl of block solution per slide for 1hr at room temperature. Make sure the block solution extends to the pap pen at the edge of the slide and completely covers the tissue to avoid uneven staining caused by surface tension near the tissue.
- Use serum from the same species in which the secondary antibody is made. Note: In this procedure, the secondary antibodies are made in donkey, and thus donkey serum is used. If secondary antibodies from two or more different species are used, include serum from each species.
- Pipette primary antibody onto slides. Note: Antibody concentrations for this staining have been optimized at 1:5,000 and 1:500 for Iba1 and Pan-neuronal, respectively. These concentrations have been found to show meaningful staining without background staining.
- Dilute the block solution to 1% serum in PBS and add primary antibodies. Pipette 300 µl of primary antibody solution in 1% serum per slide. Again, ensure the fluid is to the edge of the pap pen. Incubate overnight at 4 °C.
- Include three control slides: one that contains neither Iba1 nor Pan-neuronal antibodies, one with Iba1 without Pan-neuronal antibody, and one with Pan-neuronal antibody without Iba1. Stain these slides in the same run with the same solutions, however omit the primary antibodies to test the non-specific binding of the secondary antibodies.
- The following morning, wash slides three times in PBS for 5 min each, changing the solution between washes.
- Fluorescent antibodies are light-sensitive, therefore, from this step forward, minimize light exposure by ensuring wash containers are wrapped in foil and hybridization boxes are either black or incubated in the dark. Pipette the appropriate secondary antibodies on all slides and incubate for 60 min at room temperature at a concentration of 1:250 in block solution (see step 1.2.3) in a light-tight “humidity chamber” (see step 1.2.1).
- Use secondary antibodies of different wavelengths. Here, for the primary antibody rabbit anti-Iba1, use donkey anti-rabbit 594 as the appropriate secondary antibody. For the primary antibody mouse anti-Pan-neuronal, use donkey anti-mouse 488 as the appropriate secondary antibody. Alternatively, use anti-rabbit 488 and anti-mouse 594.
- Wash slides three times in PBS for 5 min each.
- Optional: perform nuclear staining.
- Place in Hoechst (or other nuclear stain) at a concentration of 0.03 µg/ml in double distilled H2O for exactly 60 sec.
- Wash slides three times in PBS for 5 min each.
- Wash in ddH2O.
- Coverslipping
- Coverslip slides with an aqueous mounting medium, such as Fluoromount-G or ProlongGold. Take care to remove all bubbles using a cotton-tipped applicator.
Note: Other mounting agents could be used, however high bleed-through between dyes has been noted by some within days of coverslipping. - Use clear nail polish to seal the edges, preventing the sections from drying out due to evaporation. Allow nail polish to dry in a light-tight container while slides remain flat and at room temperature, and then store in a light-tight container wrapped in foil at 4 °C.