Mar 26, 2025
Immunohistochemistry for BiPOLES(mCerulean)
This protocol is a draft, published without a DOI.
- Markowitz Lab1
- 1Georgia Institute of Technology
Protocol Citation: Markowitz Lab 2025. Immunohistochemistry for BiPOLES(mCerulean) . protocols.io https://protocols.io/view/immunohistochemistry-for-bipoles-mcerulean-d6id9ca6
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: In development
We are still developing and optimizing this protocol
Created: March 26, 2025
Last Modified: March 26, 2025
Protocol Integer ID: 125221
Disclaimer
still work in progress
Abstract
IHC for bipoles
Reagents and Tools
Reagents and Tools
Reagents
PFA
PBS
PB solution
Blocking Serum
PbTx solution
Primary Antibody
Secondary Antibody
Vectashield
Tools
Brush
12 wells plate
orbital shaker
Fixed Speed Nutating Mixer
mounting glass
cover glass
Slicing and Storing slices
Slicing and Storing slices
Post perfusion, store the brains in 4% PFA(paraformaldehyde) solution for 30-40mins then in PBS (phosphate buffer solution). Slice the brain with vibratome at desired thickness for the region of interest.
Place all the brain slices in designates 12 well plates (3 to 4 slices max in each well).
Wash
Wash
Take out the solution that brain slices were kept in 12 wells plate with 5mL transfer pipet. Place appropriate amount of PB solution (Phosphate buffer solution) in each wells and place the 12 well plates on orbital shaker ,with a lid on, for 10 mins and take out the solution with transfer pipet. Repeat this process for 3 times in total. (190rpm)
Serum Blocking
Serum Blocking
Prepare 10%[v/v] normal horse serum (NHS) in 0.3% PbTx solution (Triton X-100 containing PB solution) and place 500μL in each wells after the wash. Place the 12 wells plate on Fixed Speed Nutating Mixer for 2 hours in 4°C.
Primary Antibody Incubation
Primary Antibody Incubation
Prepare (chicken, anti-GFP, Invitorgen, A10262) at 1:500 in carrier solution (2% [v/v] NHS, in 0.3%[v/v] Triton X-100 containing PB solution). Take out the NHS solution from the wells with 5mL transfer pipet, but do not remove the solution from the negative control wells. Vortex the solution and place 500μL in each wells (besides the negative control well(s). Place the 12 wells plate back on the Fixed Speed Nutating Mixer for 48 hours in 4°C.
Wash (again)
Wash (again)
Take out the solution that brain slices were kept in 12 wells plate with 5mL transfer pipet. Place appropriate amount of PB solution (Phosphate buffer solution) in each wells and place the 12 well plates on orbital shaker ,with a lid on, for 10 mins and take out the solution with transfer pipet. Repeat this process for 3 times in total. (190rpm)
Secondary Antibody Incubation
Secondary Antibody Incubation
Take out the PB solution from all the wells and place 500μL Alexa Fluor dye-conjugated secondary antibody (goat, anti-chicken Alexa-488, Invitrogen; A11039) at 1:1000 in carrier solution (2% [v/v] NHS, in 0.3%[v/v] Triton X-100 containing PB solution). Place the 12 wells plate back on the Fixed Speed Nutating Mixer for 3 hours in 4°C.
Wash (final)
Wash (final)
Take out the solution that brain slices were kept in 12 wells plate with 5mL transfer pipet. Place appropriate amount of PB solution (Phosphate buffer solution) in each wells and place the 12 well plates on orbital shaker ,with a lid on, for 10 mins and take out the solution with transfer pipet. Repeat this process for 3 times in total. (190rpm)
Mounting
Mounting
Transfer slices onto glass slides, and mount with Vectashield with DAPI (or not with DAPI, it depends on the experiment).