May 23, 2023

Public workspaceImmunohistochemistry

DOI
dx.doi.org/10.17504/protocols.io.j8nlkwxxwl5r/v1
  • 1California Institute of Technology
Miguel Chuapoco
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkwxxwl5r/v1
Protocol Citation: Miguel Chuapoco 2023. Immunohistochemistry . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwxxwl5r/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: May 10, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 81725
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
Outlines procedures to perform immunohistochemistry on tissue that had been treated with AAV.
Protocol materials
ReagentProLong Diamond Antifade MountantThermo Fisher ScientificCatalog #P36970
Step 12
ReagentOptimal Cutting Temperature Compound (OCT)VWR InternationalCatalog #361603E
Step 5
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Step 8
ReagentNormal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121
Step 8
Tissue slicing via vibratome (mice and rhesus macaque)
Tissue slicing via vibratome (mice and rhesus macaque)
All tissue should be stored in PBS prior to slicing and further processing. Remove tissue and prepare for mounting. For mouse brain, we separate each half hemisphere. Rhesus macaque brain was separated into 4 mm thick coronal blocks. For all other tissue (e.g. liver), remove a 4-5 mm piece of tissue and cut one edge to make a flat surface.
Use super glue to mount the tissue on the provided mounting disk and fill reservoir with 1X PBS.
Equipment
Leica VT1200 S Fully automated vibrating blade microtome
NAME
Vibratome
TYPE
Leica
BRAND
VT1200S
SKU
https://www.leicabiosystems.com/us/research/vibratomes/leica-vt1200-s/
LINK

Slice tissue to 50-100 um slice and collect in 24- (mouse) or 6- (rhesus macaque) well plates filled with 1X PBS.

Tissue can also be cut to 300 um to be used for index refraction matching (e.g. to perform morphological reconstructions).
Tissue slicing via cryostat (marmoset and rhesus macaque)
Tissue slicing via cryostat (marmoset and rhesus macaque)
All tissue should be stored in PBS prior to slicing and further processing. Remove tissue and incubate in 30% sucrose at Temperature4 °C on a shaker. For marmoset tissue, incubate overnight. For rhesus macaque tissue, incubate for 72 hours, changing the 30% sucrose solution after 48 hours.

Embed tissue in OCT using 100% isopropanol (or similar) chilled with dry ice. Orient tissue to ensure coronal slices.

ReagentOptimal Cutting Temperature Compound (OCT)VWR InternationalCatalog #361603E

Equipment
Peel Away Disposable Embedding Molds
NAME
Embedding mold
TYPE
Electron Microscopy Sciences
BRAND
70182
SKU
https://www.emsdiasum.com/peel-away-disposable-embedding-molds
LINK

Equipment
Peel Away Disposable Embedding Molds
NAME
Embedding mold
TYPE
Electron Microscopy Sciences
BRAND
70184
SKU
https://www.emsdiasum.com/peel-away-disposable-embedding-molds
LINK

Equipment
Super Mega Cassette Stainless Steel Base Molds
NAME
Embedding mold
TYPE
Ted Pella Inc.
BRAND
27197-2
SKU
https://www.tedpella.com/Embedding_html/Peel-A-Way_Disposable_Histology_Molds.aspx#anchor27197
LINK

Equipment
Super Mega Cassette Stainless Steel Base Molds
NAME
Embedding mold
TYPE
Ted Pella Inc.
BRAND
27197-3
SKU
https://www.tedpella.com/Embedding_html/Peel-A-Way_Disposable_Histology_Molds.aspx#anchor27197
LINK

Store tissue at Temperature-80 °C overnight before continuing to slicing.

Cut tissue to 50 um slice and collect in 24- (marmoset) or 6- (rhesus macaque) well plates filled with 1X PBS.
Immunohistochemistry
Immunohistochemistry
Incubate slices in primary antibody overnight on a shaker at room temperature. Dilute antibody in 1X PBS supplemented with 0.1% Triton X-100 ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML and 10% normal donkey serum ReagentNormal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121 .

ABCDE
Antibody epitopeVendorCat #RRIDDilution
GLUT1Millipore-Sigma07-1401AB_15870741:200
HACell Signaling Technology3724AB_15495851:200
GFPAves LabsGFP-1020AB_100002401:500
NeuNAbcamab177487AB_25321091:200
S100 Abcamab52642AB_8824261:200

Wash with PBS 3-5 times at room temperature on a shaker.
Incubate slices in secondary antibody overnight on a shaker at room temperature. Dilute antibody in 1X PBS supplemented with 0.1% Triton X-100 and 10% normal donkey serum.

Protect from light to avoid photobleaching.


ABCDE
Antibody epitopeVendorCat #RRIDDilution
rabbit IgGJackson Immunoresearch Labs711-605-152AB_24922881:200
chicken IgYJackson Immunoresearch Labs703-605-155AB_23403791:200

Wash with PBS 3-5 times at room temperature on a shaker.

Protect from light to avoid photobleaching.

Mount sections on slides and allow slice to dry at room temperature in the dark. Mount coverslip using Prolong Diamond Antifade. ReagentProLong Diamond Antifade MountantThermo Fisher ScientificCatalog #P36970