Jul 06, 2024

Public workspaceImmunohistochemistry for brain sections

Science Advances
DOI
dx.doi.org/10.17504/protocols.io.3byl4956jgo5/v1
  • 1Department of Neurosurgery, Weill Cornell Medical College, New York, NY 10065;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Eileen Ruth Torres
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DOI: dx.doi.org/10.17504/protocols.io.3byl4956jgo5/v1
External link: https://doi.org/10.1126/sciadv.adn6016
Protocol Citation: Santiago Unda, Roberta Marongiu, Michael G. Kaplitt 2024. Immunohistochemistry for brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4956jgo5/v1
Manuscript citation:
Kokotos AC, Antoniazzi AM, Unda SR, Ko MS, Park D, Eliezer D, Kaplitt MG, Camilli PD, Ryan TA Phosphoglycerate kinase is a central leverage point in Parkinson’s disease–driven neuronal metabolic deficits. Science Advances 10(34). doi: 10.1126/sciadv.adn6016
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: July 06, 2024
Last Modified: July 06, 2024
Protocol Integer ID: 102917
Keywords: ASAPCRN, immunohistochemistry, brain, mouse
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
Protocol for processing 30um mouse brain sections for immunolabeling.
Materials
Solutions:
TBS-T
For 2l:
50mM Tris pH 7.5 – 100 ml of 1M stock
150 mM NaCl – 17.53 g
Triton 0.2 % - 4 ml
DW – 1.9 l
ANTI-FREEZE SOLUTION
For 1l:
400 ml PBS/TBS (no TritonX-100!)
300 ml ethylene glycol
300 ml glycerine
STIR SOLUTION!!
Blocking solution
Prepare the solution in falcon tubes:
10 % goat, donkey or horse serum in TBS-T
Attn.: Do not shake too much (mix by inverting only). Proteins generate bubbles. Between experiments keep the blocking solution in the refrigerator at Temperature4 °C . During experiment keep TemperatureOn ice .

Tissue Preparation
Tissue Preparation
Sections at cryostat:
  • Thickness 30 µm
  • Sections picked up with a wet brush (not too wet) and moved to the TBS-T wells (usually serial sections). Store section in anti-freeze solution in Temperature-20 °C if not immediately staining.

Transfer the sections into the net-wells.
Day 1: Primary Antibody Incubation
Day 1: Primary Antibody Incubation
Washes: 3-step wash, Duration00:10:00 each, on a shaker, in TBS-T. (Between washes, just move the inserts from one tray to another.)
10m
Blocking step: add blocking solution into a 6-well plate.
Transfer the sections with a clean brush from the net-inserts into the 6-well plate.
Incubate on the shaker for Duration01:00:00 at TemperatureRoom temperature .
1h
Primary antibody: Transfer the sections into a 6-well plate containing the primary antibody solution (1 ml per well).
Incubation: on a shaker in the cold room DurationOvernight .
1h
Day 2: Secondary Antibody Incubation
Day 2: Secondary Antibody Incubation
1h
1h
Washes: a 3-step wash, Duration00:10:00 each, in TBS-T.
10m
Secondary antibody: Transfer the sections into a 12-well plate containing the secondary antibody. The secondary antibody will be incubated atTemperatureRoom temperature .
Incubation: on a shaker, at TemperatureRoom temperature , for 1-2 hours.
Washes: 3-4 times, for Duration00:10:00 , in TBS-T.
10m
DAPI: incubate with DAPI (1:10,000) shaking forDuration00:10:00 at TemperatureRoom temperature .
10m
Washes: a 2-step wash, on a shaker, atTemperatureRoom temperature , Duration00:10:00 each.
10m
Mounting: Mount sections and let them dry for Duration00:10:00 atTemperatureRoom temperature .
10m
Coverslip: Let the slides dry at least DurationOvernight at TemperatureRoom temperature .
10m