FREE-FLOATING IMMUNOHISTOCHEMISTRY PROTOCOL



FREE-FLOATING IMMUNOHISTOCHEMISTRY PROTOCOL (Chromogen development)

June 2018

All steps are done with gentle agitation on a rotating platform. All steps except for antibody and A+B incubations are done in Corning Netwell inserts which we use for staining of free-floating tissue sections (6- or 12 well, 74 um mesh). Antibody incubations (primary and secondary) and Vectastain A+B steps are done without nets by moving sections into 12 or 24 well plates, depending on the number of sections to be stained, which require only 500-800 ul (24 well) or 1 ml (12 well) of solution, respectively. Use a fine paint brush (#0 or #1) to move sections between nets and wells, with utmost care!

1. Wash sections several times in TBS to remove antifreeze.

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 (DAB).

3. If using Vectastain ABC (HRP-DAB) for development, quench endogenous peroxidase activity by incubating sections in 0.9% hydrogen peroxide in TBS with 0.01% Triton X-100 for 30 min at RT.

*Alternatively, endogenous peroxidase activity can be quenched in 0.3% hydrogen peroxide in 100% methanol or with up to 3% hydrogen peroxide in TBS plus 10% methanol.

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.

*Alternatively, antigen retrieval solution can be pre-warmed in glass dish using a 90º C water bath. Transfer sections to heated solution in water bath for 10 minutes. Remove glass dish with sections from water bath and allow to cool to RT.

6. Block for 1-2 hr at RT with 3-10% serum in TBS-T (TBS plus 0.1% Triton-X 100). When using Vectastain ABC kits for secondary, use the serum that comes with the kit for blocking at the recommended 1.5% serum concentration. Use 12 or 24 well plates for antibody incubation to decrease the amount of reagent required. Add sections one at a time to wells to ensure all are moving freely in solution.

*Optional - Triton can be increased 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.

*Optional - add BSA at 0.5-1% to serum-containing block to decrease non-specific binding of primary.

7. Incubate in primary antibody diluted as needed in blocking solution overnight at 4° C. Alternatively, NSALabs and Vector Labs both suggest using TBST for primary dilutions and incubating free floating sections at RT overnight. Some antibodies (i.e. parvalbumin, caspase, fractin) 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 12 or 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 in block or TBST for 1-2 hr at RT. For Vectastain ABC, be sure to use the serum and antibody provided in the kit; secondary can be diluted up to 1:500 and incubated 1-2 hr at RT or overnight at 4º C. Use 12 or 24 well plates for antibody incubation to decrease the amount of reagent used.

10. Return sections to nets and wash 3 x 10 min with TBS. If using Vectastain kits, mix parts A + B in TBS (1-2 drops each per 10 ml of TBS) during the first wash and let sit for 30 min at RT. If using fluorescently labeled secondary, go to step 15.

11. Incubate sections in A+B for 30-90 min at RT. Use 12 or 24 well plates for antibody incubation to decrease the amount of reagent used.

12. Return sections to nets and wash 3 x 10 min with TBS.

13. Develop chromagen in DAB solution for desired time, which must be determined empirically. For Sigmafast D4418 tablets, dissolve 1 of each tablet into 50 ml water and filter before use. Deactivate with bleach before disposing in chemical waste.

*Alternatively, use DAB from stock solutions. For 100 ml: add 1-2 ml of 10 mg/ml DAB stock or Sigma D5905 tablets, 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.

14. Mount sections from 0.05 M sodium phosphate or 0.1 M sodium nitrate (without 0.9% NaCl) onto Superfrost Plus slides. At a minimum, let slides dry overnight. Rehydrate in water, then dehydrate/defat through alcohol series (70%, 95%, 100%) into xylene and coverslip with Permount, or else rehydrate back to water and counterstain with hematoxylin if desired. Leave upright overnight in slide holder for drying to allow excess Permount to flow out.

*If sections are not well fixed or do not adhere well to the slide, skip water rehydration and go directly through alcohol series and into xylene.

FREE-FLOATING IMMUNOFLUORESCENCE PROTOCOL

June 2018

All steps are done with gentle agitation on a rotating platform. All steps except for blocking and antibody incubations are done in Corning Netwell inserts which we use for staining of free-floating tissue sections (6- or 12 well, 74 um mesh). Antibody incubations (primary and secondary) are done without nets by moving sections into 12 or 24 well plates, depending on the number of sections to be stained, which require only 500-800 ul (24 well) or 1 ml (12 well) of solution, respectively. Use a fine paint brush (#0 or #1) to move sections between nets and wells, with utmost care!

1. Wash sections several times in TBS to remove antifreeze.

2. Block for 1-2 hr at RT with 3-10% serum in TBS-T (TBS plus 0.1% Triton-X 100). Add sections one at time to wells or else be sure to give the net a good jiggle to ensure the sections are moving freely. Use 12 or 24 well plates to decrease the amount of reagent required.

*Optional - Triton can be increased 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.

*Optional - add BSA at 0.5-1% to serum-containing block to decrease non-specific binding of primary.

3. Incubate in primary antibody diluted as needed in blocking solution or in TBS-T overnight at 4° C or RT. Some antibodies (i.e. parvalbumin, caspase, fractin) 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 12 or 24 well plates for antibody incubation to decrease the amount of reagent required.

4. Return sections to nets and wash 3 x 10 min with TBS.

5. Incubate in secondary antibody diluted 1:500 in blocking solution 1-2 hr at RT or overnight at 4° C. Use 12 or 24 well plates for antibody incubation to decrease the amount of reagent required. PROTECT FROM LIGHT.

6. Return sections to nets and wash 3 x 10 min with TBS. Keep covered to protect from light.

7. If needed, incubate in tertiary antibody diluted 1:500 in blocking solution overnight at 4° C. This step can help to increase signal from weak antigens such as fractin and caspase 3 if the same fluorophore is used for secondary and tertiary antibodies (ie, Alexa 594 on both a Gt anti-rabbit and Dk anti-goat). Following incubation, return sections to nets and wash 3 x 10 min with TBS. Keep covered to protect from light.

8. Mount sections from TBS onto SuperFrost Plus slides. Do not let dry - coverslip while still wet using antifade reagent such as Vectashield Hardset from Vector Labs or ProLong Gold/ProLong Diamond from Thermo. Leave upright on a drying rack to allow excess mounting medium to flow out. Keep covered to protect from light.

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