Apr 24, 2023
IHC_cFOS+Parvalbumin
- 1Wesleyan University
Protocol Citation: Bilge Buyukdemirtas 2023. IHC_cFOS+Parvalbumin. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyb9yvpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2023
Last Modified: May 31, 2023
Protocol Integer ID: 80981
Abstract
Immunohistochemistry protocol for c-FOS and Parvalbumin co-staining of mouse brain tissue slices (30 microns thick).
Guidelines
All shaker incubations are done at 200 rpm speed.
Staining
Staining
Antigen Retrieval
Remove PBS from wells. Add 500ul 0.3% citrate buffer into each well.
Incubate at RT on shaker for 30 min
Incubate at 65 °C for 45 min
Cool down on the bench for 20 min
3X PBS Washes
Remove citrate buffer from wells and replace with 500ul 1X PBS (or use a dropper pipette and fill the well halfway)
Incubate at RT on shaker for 15 min remove PBS and replace with new 1X PBS, repeat two more times
Block Non-Specific Signal
Make enough: 10% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS for all samples (500ul per well)
Remove PBS and replace with blocking reagent 500ul per well.
Incubate on shaker RT for 60min.
Primary Antibody
Make enough: mouse anti-parv (1:1000, Sigma-Aldrich, #P3088) and rabbi anti-cfos (1:1000, cell Signaling, #2250S) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
Replace the blocking reagent with primary antibody mix, 500 ul per well, and incubate at 4 °C
Overnight
DAY 2
3X PBS Washes
Remove primary Ab mix from wells and replace with 500ul 1X PBS (or use a dropper pipette and fill the well halfway)
Incubate at RT on shaker for 15 min repeat two more times
Secondary Antibody
Make enough: goat anti-mouse AF-555 (1:500, Invitrogen, #A-21422) and biotin goat anti-rabbit (1:1000, Vector Labs, #PK-6101) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
Remove PBS from wells and replace with 500ul Secondary antibody mix. Cover with aluminum foil. Note: secondary antibodies are light sensitive, so from this point on the plate MUST be covered from light as much as possible.
Incubate at RT on shaker for 120 min.
3X PBS washes as described in Step 5 and 5.1 with the plate covered
Tertiary Antibody (for cFOS only)
make enough: AF-488 streptavidin conjugated (1:1000, Thermo Fisher, #S11223) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
Replace PBS in wells with 500uL of tertiary antibody solution, cover the plate with aluminum foil, and incubate at RT on shaker for 120 min.
3X PBS washes as described in step 5 and 5.1 with the plated covered
Mounting with medium with and without DAPI