FREE-FLOATING IMMUNOHISTOCHEMISTRY PROTOCOL
1. Wash sections several times in TBS to remove antifreeze. All steps are done with gentle agitation on a rotating platform. All steps except for antibody and A+B (Vectastain only) incubation are done in nets.
2. If staining for amyoid, pre-treat with 80-100% formic acid for 1 minute, then skip peroxide quenching and go to step 4.. If staining for anything else, skip this step and go to step 3.
3. If using Vectastain ABC (HRP-DAB) for development, quench endogenous peroxidase activity by incubating sections in either 0.3% hydrogen peroxide in 100% methanol or with up to 3% hydrogen peroxide in TBS plus 10% methanol or with 0.6% peroxide in TBS without methanol for 30 min at RT. If using a fluorescently tagged secondary, skip to step 4.
4. Wash 3 x 10 min with TBS.
5. Some antigens require antigen retrieval for antibody detection. We generally use 10 mM sodium citrate pH 6.0 for this. Heat the solution to boiling in a microwave oven, transfer the sections into the boiling solution and let cool to RT (approx. 30 min). After cooling, wash 3 x 5 min with TBS.
6. Block for 1-2 hr at RT with 3-10% serum in TBS plus 0.1% Triton-X 100. When using Vectastain ABC kits for secondary, use the serum that comes with the kit for blocking and reduce concentration to the recommended 1.5%. Optional - increase Triton to 0.4% during blocking step to better permeabilize cells. The permeabilization step can also be done separately prior to blocking as an additional incubation with 0.4% Triton in TBS for 30 min. Another optional component for block buffer is BSA at 0.5-1% - this can be added to serum-containing block to decrease non-specific binding of primary.)
7. Incubate in primary antibody diluted as needed in block (3-10% serum with 0.1% Triton X 100 in TBS) or in TBST (TBS + 0.1% Triton without serum) overnight at 4° C or RT. Hopkins Neuropath regularly diluted primaries in block and incubated paraffin sections at RT or free-floating sections at 4º C overnight. Based on tips from NSA Labs and Vector Labs, we are now using TBST for our primary dilutions and incubating free floating sections at RT overnight. Some antibodies (i.e. parvalbumin) do best when left for 72 hr at 4º C. Optimal conditions for each antibody will need to be worked out by trial and error. Use 24 well plates for antibody incubation to decrease the amount of reagent required.
8. Return sections to nets and wash 3 x 10 min with TBS.
9. Incubate in secondary antibody diluted as needed in block (3-10% serum with 0.1% Triton X 100 in TBS) 1-2 hr at RT. For Vectastain ABC, make sure to use the serum provided in the kit; secondary can be diluted up to 1:500. Use 24 well plates for antibody incubation to decrease the amount of reagent used.
10. Wash 3 x 10 min in TBS. If secondary is fluorescently-tagged, go to step 14. If using a Vectastain ABC kit for HRP-DAB development, mix parts A and B in TBS at this point and let sit for 30 min at RT. Dilutions of A and B can be halved from the manufacturer's recommendation and still work well (1:100 rather than 1:50 each).
11. Incubate sections in A+B for 90 min at RT.
12. Wash 3 x 10 min in TBS.
13. Develop chromagen in DAB solution (for 100 ml add 1-2 ml of 10 mg/ml stock or Sigma D5905 tablets (can be increased from 0.1 up to 0.25 mg/ml), 100 ul of 1% NiCl (can be increased up to 500 ul of 8% stock), 30 ul of 30% hydrogen peroxide in 97 ml TBS). Filter before use. Deactivate with bleach before disposing.
14. Mount sections from 0.05 M sodium phosphate or 0.1 M sodium nitrate (without 0.9% NaCl) onto Superfrost Plus slides. Let dry overnight. Rehydrate in water, then dehydrate/defat through alcohols and into xylene. Rehydrate to water and counterstain with hematoxylin if desired. Coverslip with Permount.
15. For fluorescent secondaries, after washing in TBS, mount sections onto SuperFrost Plus slides. Do not let dry - coverslip while still wet using Antifade reagent such as Aquamount from Polysciences or Vectashield from Vector Labs. Lay flat to dry, protect from light.