TITLE: Immunohistochemistry (IHC) on mouse brain slices
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
This protocol provides a starting point for immunohistochemical localization and visualization of proteins in mouse brain tissue. This can be useful for marking cell types of interest or investigating the effect of experimental manipulations on protein expression and/or localization. 

This protocol is compatible with free-floating tissue or tissue that has already been mounted onto a slide. 

[BEFORE_START]
This protocol assumes that you are starting from free-floating brain slices in 1x PBS, or from sections mounted on a glass slide. 

We typically process free-floating sections 30 µm to 100 µm thick, and mounted sections 5 µm  to 100 µm thick. 

Free-floating sections may allow for better penetration of antibodies into thick sections (as diffusion occurs from both sides). Mounting sections onto a slide may be more convenient for some sectioning methods (cryosectioning) and can enable thinner sections to be collected without risk of damaging sections during processing. Depending on the size of well-plate used, mounted sections may use less antibody, and may thus be cheaper to process. Mounting sections on slides is also recommended if incorporating an antigen retrieval step.

[STEPS]
SECTION: Reagent set up
1. General note on reagents and consumables

 

SECTION: Reagent set up
2. Prepare 1x PBS, by combining: 

1 PBS packet
MilliQ water (or equivalent): 1 L 

Shake to combine and store at Room temperature for up to 3 months.

(Optional) Treat 1x PBS with DEPC- or DMPC-treat to inactivate RNases. Add 1 mL of DEPC or DMPC to 1 L of 1x PBS, shake with lid tightly closed, then loosen lid and let sit   (or if needed same day, for 120 min at 37 °C ). After incubation, autoclave the 1x PBS to inactivate the DEPC or DMPC. Allow to cool to room temperature before using. 

 

SECTION: Reagent set up
3. Prepare blocking / antibody dilution buffer. First determine how much will be needed. For every well, you will need 1.5 mL . For every slide, you will need 1 mL . This blocking buffer will be used for blocking, primary antibody dilution, and secondary antibody dilution. Make extra (~10%) to ensure there is enough for each well. 

Example: for 6 wells with free-floating sections, prepare 10 mL of blocking buffer, by combining:

BlockAid Blocking Solution: 9.9 mL 
10% Triton X-100: 100 µL 

Prepare on first day of protocol, and store at 4 °C until use.

 

SECTION: Immunohistochemistry on mouse brain sections
4. This protocol diverges depending on whether you are processing free-floating sections or mounted sections. If you are incorporating an antigen retrieval step, we recommend using slide mounted sections or mount free-floating sections on a slide before proceeding. 

Once samples have some fluorescence, keep samples in the dark. E.g. if samples have endogenous fluorescence (e.g. GFP or tdTomato-expression), keep samples in dark through the entire protocol. If fluorescence is introduced with secondary antibody, keep samples in dark during and after secondary antibody incubation. 
SECTION: Immunohistochemistry on mouse brain sections
7. If necessary, transfer the desired slices to a new well plate filled with 1x PBS. Use a paintbrush to transfer sections. 
SECTION: Immunohistochemistry on mouse brain sections
5. Note on scaling and volumes

 

SECTION: Immunohistochemistry on mouse brain sections
8. Aspirate and dispose of 1x PBS in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Add 500 µL of blocking buffer to each well. Incubate for 60 min at Room temperature with gentle shaking.
SECTION: Immunohistochemistry on mouse brain sections
6. Note on serial sections

 

SECTION: Immunohistochemistry on mouse brain sections
5. Note on scaling and volumes

 

SECTION: Immunohistochemistry on mouse brain sections
9. In last 5 minutes of the blocking step, prepare the primary antibody solution. Dilute the primary antibody in blocking buffer and mix by inverting gently. 

 

SECTION: Immunohistochemistry on mouse brain sections
10. Once blocking is complete, aspirate and dispose of blocking buffer in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Add 500 µL of primary antibody dilution to each well. Incubate.    at 4 °C  with gently shaking. 

 

SECTION: Immunohistochemistry on mouse brain sections
11. Following primary antibody incubation, aspirate and dispose of primary antibody solution in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Wash samples 3 times, with 1 mL of 1x PBS per wash. Each wash should be 15 min at Room temperature , with gentle shaking. 

 

SECTION: Immunohistochemistry on mouse brain sections
12. During last 5 minutes of washes, prepare secondary antibody solution. Dilute the secondary antibody in blocking buffer and mix by inverting gently. 

 

SECTION: Immunohistochemistry on mouse brain sections
13. Once washes are done, aspirate and dispose of 1x PBS in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Add 500 µL of secondary antibody dilution to each well. Incubate for 60 min at 4 Room temperature  with gently shaking. 
SECTION: Immunohistochemistry on mouse brain sections
14. Following secondary antibody incubation, aspirate and dispose of secondary antibody solution in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Wash samples 3 times, with 1 mL of 1x PBS per wash. Each wash should be 15 min at Room temperature , with gentle shaking. 
SECTION: Immunohistochemistry on mouse brain sections
15. (Optional) Stain sections with Hoechst 33342 or DAPI to label nuclei. For each well, prepare 500 µLof 1x PBS with 1/10000 Hoechst 33342 or DAPI. 

Aspirate and dispose of 1x PBS in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Add 500 µL of Hoechst or DAPI solution. Incubate 15 min at Room temperature with gentle shaking. 

Following incubation with Hoechst or DAPI solution, aspirate and dispose of Hoechst or DAPI solution in wells. Use the same pipette tip for each well, but be careful not to transfer tissue between wells. Ensure complete removal.

Wash samples 3 times, with 1 mL of 1x PBS per wash. Each wash should be 5 min at Room temperature , with gentle shaking. 
SECTION: Immunohistochemistry on mouse brain sections
16. Mount sections on slide. Use paintbrush to transfer and flatten sections onto surface of slide. Once all sections are placed, excess 1x PBS can be removed with a Kimwipe and/or by placing slide in a fume hood. Allow sections to dry on slide (sections are dried once the tissue appears transparent). 

Once tissue is dry, pipette 100 µL  of mounting media in a line on the surface of the slide, near the tissue sections. Spread the mounting media over the tissue by gently lowering a clean coverslip over the tissue, being careful not to introduce bubbles. 

Once all coverslips are mounted, gently press down on each coverslip to push out excess mounting media. Store in the dark at Room temperaturefor at least   until mounting media cures. Once cured, excess Prolong Diamond can be easily removed by running slides under a gentle stream of MilliQ water, being careful to not displace coverslips. 

 

SECTION: Immunohistochemistry on mouse brain sections
6. If mounting free-floating sections onto slides:

Use paintbrush to transfer and flatten sections onto surface of slide. Once all sections are placed, excess 1x PBS can be removed with a Kimwipe and/or by placing slide in a fume hood. Allow sections to dry on slide (sections are dried once the tissue appears transparent). Once dried, place slides in a Coplin jar filled with 1x PBS. 

If starting from sections already mounted on a slide:

Remove slide box from storage and allow to warm to room temperature before opening. If necessary, wash excess O.C.T. from slides by placing tissue in a Coplin jar filled with 1x PBS, incubate 5 min at Room temperature and then decant 1x PBS from Coplin jar and refill with fresh 1x PBS. 
SECTION: Immunohistochemistry on mouse brain sections
8. Remove slides from Coplin jar and lean against a vertical surface for 30 s  . Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled 1x PBS. Dry the back of the slides with a Kimwipe, then transfer to a staining tray with tissue side up. 

Pipette 600 µL of blocking buffer onto the surface of the slide, ensuring that all the tissue is covered. Incubate for 60 min at Room temperature . 
SECTION: Immunohistochemistry on mouse brain sections
9. In last 5 minutes of the blocking step, prepare the primary antibody solution. Dilute the primary antibody in blocking buffer and mix by inverting gently. 

 

SECTION: Immunohistochemistry on mouse brain sections
10. Once blocking is complete, decant blocking buffer from slides. Lean slide against a vertical surface for 30 s. Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled blocking buffer. Transfer back to a staining tray with tissue side up. 

Add 200 µL of primary antibody dilution to each slide. Spread the primary antibody solution over the tissue by gently lowering a clean coverslip or appropriately sized piece of parafilm over the tissue, being careful not to introduce bubbles. 

Incubate    at 4 °C .

 

SECTION: Immunohistochemistry on mouse brain sections
11. Following primary antibody incubation, remove coverslips or parafilm by lifting and holding the slide vertically, allowing the coverslip or parafilm to slide off. Lean slide against a vertical surface for 30 s. Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled primary antibody solution. 

Transfer slides to Coplin jar and wash 3 times with 1x PBS. When switching wash buffers, decant old wash buffer into a liquid waste collection beaker, using a finger to keep slides in the jar, then add new wash buffer, being careful not to pour PBS directly onto the tissue sections. Each wash should be 15 min at Room temperature , with gentle shaking. 

 

SECTION: Immunohistochemistry on mouse brain sections
12. During last 5 minutes of washes, prepare secondary antibody solution. Dilute the secondary antibody in blocking buffer and mix by inverting gently. 

 

SECTION: Immunohistochemistry on mouse brain sections
13. Once washes are done, remove slides from Coplin jar and lean against a vertical surface for 30 s  . Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled 1x PBS. Dry the back of the slides with a Kimwipe, then transfer to a staining tray with tissue side up. 

Add 200 µL of secondary antibody dilution to each slide. Spread the secondary antibody solution over the tissue by gently lowering a clean coverslip or appropriately sized piece of parafilm over the tissue, being careful not to introduce bubbles. Incubate for 60 min at 4 Room temperature .
SECTION: Immunohistochemistry on mouse brain sections
14. Following secondary antibody incubation, remove coverslips or parafilm by lifting and holding the slide vertically, allowing the coverslip or parafilm to slide off. Lean slide against a vertical surface for 30 s. Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled secondary antibody solution. 

Transfer slides to Coplin jar and wash 3 times with 1x PBS. When switching wash buffers, decant old wash buffer into a liquid waste collection beaker, using a finger to keep slides in the jar, then add new wash buffer, being careful not to pour PBS directly onto the tissue sections. Each wash should be 15 min at Room temperature , with gentle shaking. 
SECTION: Immunohistochemistry on mouse brain sections
15. (Optional) Stain sections with Hoechst 33342 or DAPI to label nuclei. For each slide, prepare 500 µLof 1x PBS with 1/10000 Hoechst 33342 or DAPI. 

Remove slides from Coplin jar and lean against a vertical surface for 30 s  . Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled 1x PBS. Dry the back of the slides with a Kimwipe, then transfer to a staining tray with tissue side up. 

Add 500 µL of Hoechst or DAPI solution to surface of each slide ensuring that all the tissue is covered. Incubate for 15 min at Room temperature . 

Once Hoechst or DAPI staining is complete, decant staining buffer from slides. Lean slide against a vertical surface for 30 s. Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled Hoechst or DAPI solution.

Transfer slides to Coplin jar and wash 3 times with 1x PBS. When switching wash buffers, decant old wash buffer into a liquid waste collection beaker, using a finger to keep slides in the jar, then add new wash buffer, being careful not to pour PBS directly onto the tissue sections. Each wash should be 5 min at Room temperature , with gentle shaking. 
SECTION: Immunohistochemistry on mouse brain sections
16. Dry sections on slides and mount coverslip. 

Remove slides from Coplin jar and lean against a vertical surface for 30 s  . Gently tap bottom of slide against Kimwipe or a piece of paper towel on the bench surface to remove pooled 1x PBS. Dry the back of the slides with a Kimwipe, then transfer to a bench top or fume hood to dry (sections are dried once the tissue appears transparent). 

Once tissue is dry, pipette 100 µL  of mounting media in a line on the surface of the slide, near the tissue sections. Spread the mounting media over the tissue by gently lowering a clean coverslip over the tissue, being careful not to introduce bubbles. 

Once all coverslips are mounted, gently press down on each coverslip to push out excess mounting media. Store in the dark at Room temperaturefor at least   until mounting media cures. Once cured, excess Prolong Diamond can be easily removed by running slides under a gentle stream of MilliQ water, being careful to not displace coverslips. 

 

SECTION: Immunohistochemistry on mouse brain sections
7. (Optional) Perform antigen retrieval. 

Dilute 10x Citrate buffer, pH 6. For one Coplin jar filled with slides, 50 mL should be adequate:

10x Citrate buffer, pH 6: 5 mL 
UltraPure water: 45 mL 

Transfer slides to a new Coplin jar filled with 1x Citrate buffer, and boil for 10 min  in microwave. Allow Coplin jar to cool at Room temperature for 30 min . Decant antigen retrieval buffer from Coplin jar and wash 3 times with 1x PBS. When switching wash buffers, decant old wash buffer into a liquid waste collection beaker, using a finger to keep slides in the jar, then add new wash buffer, being careful not to pour PBS directly onto the tissue sections. Each wash should be 15 min at Room temperature , with gentle shaking. 

Transfer back to original Coplin jar, filled with 1x PBS.