Jul 13, 2022

Public workspaceImmunohistochemistry

DOI
dx.doi.org/10.17504/protocols.io.b9ubr6sn
  • Alexandra Nelson1
  • 1University of California San Francisco
Kelsey Barcomb
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DOI: dx.doi.org/10.17504/protocols.io.b9ubr6sn
Protocol Citation: Alexandra Nelson 2022. Immunohistochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.b9ubr6sn
Manuscript citation:
Jonathan S Schor, Isabelle Gonzalez Montalvo, Perry WE Spratt, Rea J Brakaj, Jasmine A Stansil, Emily L Twedell, Kevin J Bender, Alexandra B Nelson (2022) Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in Parkinsonian mice eLife 11:e75253

https://doi.org/10.7554/eLife.75253
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: May 23, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63075
Keywords: Immunohistochemisty, IHC, Immunostaining, Brain, Mouse, ASAPCRN
Abstract
This protocol describes immunohistochemical staining of fixed brain sections.
Sectioning
Using freezing microtome section brain at 30-35 μm
Place sections in PBS in a 24-well plate until ready for immunohistochemistry; if not performing immunohistochemistry that day, cover 24 well plate and place at 4ºC overnight
Blocking.
Incubate sections in blocking buffer at TemperatureRoom temperature for 1-2 hours.
Blocking buffer is: 3% NDS (normal donkey serum)/ 0.1% triton (also called NDST) in PBS.
Primary Antibody.
Prepare primary antibody in 3% NDS at desired concentration (make sure this is the final concentration in the well if you are adding more than one primary) and let sections sit on shaker DurationOvernight at Temperature4 °C

Wash.
Wash sections 3-5 times in PBS for 10-15 minutes each at TemperatureRoom temperature

Secondary Antibody.
Prepare secondary antibody in 3% NDS at desired concentration and let sections sit on shaker for 2-4 hours at Temperature4 °C
Note
1:500 is default concentration for secondary antibodies.



Wash.
Wash sections 3-5 times in PBS for 10-15 minutes each at TemperatureRoom temperature
Mounting.
Mount sections onto slides with Vectashield hardset mounting medium (with or without DAPI).
Imaging.
For low-magnification display of viral expression patterns or tyrosine hydroxylase staining, we typically use 10X stitched epifluorescence images.
For evaluation of individual neurons and overlap of fluorescent markers, we typically use 40X epifluorescence or confocal Z-stacks.