Jul 06, 2022

Public workspaceExpression of molecular markers in mouse and human stellate ganglia

DOI
dx.doi.org/10.17504/protocols.io.rm7vzyko5lx1/v1
  • 1Columbia University Irving Medical Center;
  • 2Naomi Berrie Diabetes Center, Division of Molecular Genetics, Columbia University Irving Medical Center, New York, United States;
  • 3Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, United States
Daniele Neri
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzyko5lx1/v1
Protocol Citation: Daniele Neri, Seoeun Lee, Alexandre J Lafond, Lori Zeltser 2022. Expression of molecular markers in mouse and human stellate ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzyko5lx1/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 01, 2022
Last Modified: July 06, 2022
Protocol Integer ID: 60202
Abstract
Protocol for immunohistochemistry
Materials
Equipement:
Equipment
Fisherbrand™ Superfrost™ Plus Microscope Slides
NAME
Microscope Slides
TYPE
Fisherbrand
BRAND
12-550-15
SKU
https://www.fishersci.com/shop/products/fisherbrand-superfrost-plus-microscope-slides-2/12-550-15?searchHijack=true&searchTerm=fisherbrand-superfrost-plus-microscope-slides-2&searchType=Rapid&matchedCatNo=12-550-15
LINK
For frozen sections, cytocentrifuge preparations, and standard Papanicolaou smears Frosted: ¾” SuperFrost Tab Type: Microscope Slide Width (Metric): 25 mm Corner Style: Regular Length (Metric): 75 mm Material: Glass Color: White
SPECIFICATIONS

Equipment
Fisherbrand™ Cover Glasses: Rectangles
NAME
Cover Glasses
TYPE
Fisherbrand
BRAND
12-544-EP
SKU
https://www.fishersci.com/shop/products/cover-glasses-rectangles-promo-22/12-544-EP?searchHijack=true&searchTerm=cover-glasses-rectangles-promo-22&searchType=Rapid&matchedCatNo=12-544-EP
LINK
Material: Borosilicate Glass Length (Metric): 50 mm Dimensions (L x W): 50 x 24 mm For Use With: Microscope, Histology, Cytology, Hematology, Microbiology Product Type: Coverglass No. 1.5 Shape: Rectangular Width (Metric): 24 mm Thickness (Metric): 0.16 to 0.19 mm
SPECIFICATIONS

Equipment
ImmEDGE Hydrophobic Barrier Pen
NAME
Pen
TYPE
Vector Laboratories
BRAND
NC9545623
SKU
https://www.fishersci.com/shop/products/immedge-pen/NC9545623
LINK
Applications: Immunohistochemistry / Immunocytochemistry, Immunofluorescence, In situ hybridization
SPECIFICATIONS
Reagents:


Reagent16% Paraformaldehyde Aqueous SolutionFisher ScientificCatalog #50-980-488
ReagentPhosphate Buffered SalineFisher ScientificCatalog #BP3994
ReagentVECTASHIELD® Hardset™ Antifade Mounting MediumVector LaboratoriesCatalog #H-1400
ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML
ReagentAndwin Scientific Tissue-Tek™ CRYO-OCT CompoundFisher ScientificCatalog #14-373-65
ReagentDAPIFisher ScientificCatalog #D3571
ReagentNormal Donkey SerumJackson ImmunoresearchCatalog #017-000-121

Primary Antibodies:
ABCDE
ReagentDilutionProviderCat. No/SKURRID
Anti-Tyrosine Hydroxylase Antibody1:100Millipore-SigmaAB152 AB_390204
Anti-Tyrosine Hydroxylase Antibody1:400Abcamab76442AB_1524535
Anti-Neuropeptide Y Antibody1:500Sigma-AldrichM9528AB_260814
Anti-Vasoactive Intestinal Peptide Antibody1:500Immunostar20077AB_572270
Secondary Antibodies:
ABCDE
ReagentDilutionProviderCat. No/SKURRID
Anti-Rabbit Secondary Antibody1:500ThermoFisher ScientificA11034AB_2576217
Anti-Rabbit Secondary Antibody1:500ThermoFisher ScientificA31572AB_162543
Anti-Chicken Secondary Antibody1:500ThermoFisher ScientificA21449AB_1500594



Before start
The human stellate ganglia (SG) were provided by Dr. Olujimi Ajijola at UCLA. The tissue was collected post-mortem from a 44-year-old female donor and dissected immediately after the donor's death.
The dissection procedure of murine SG is described in the "Tissue Harvesting" section of our protocol, "Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection."
Protocol
Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection
NAME

Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection

CREATED BY
Daniele Neri

Postfixation of the tissue
Postfixation of the tissue
Both mouse and human stellate ganglia (SG) were fixed in 4% PFA in 0.1M PB overnight at 4°C, washed with Phosphate Buffer Saline (PBS) 4°C for at least 1 h
Encasing and slicing
Encasing and slicing
Ganglia were encased in Optimal Cutting Temperature compound (Tissue-Tek) cut into 10 μm cryo-sections on slides, and stored at -80°C

Immunohistochemistry
Immunohistochemistry
Thaw the sections at room temperature for at least 1 hour
Wash in PBS for 30 min twice at room temperature
Incubate in blocking buffer (5% donkey serum in PBS with 0.1% Triton X-100) for 1 hour at room temperature
Incubate the sections with primary antibodies diluted in blocking buffer overnight at 4C
Wash in PBS for 30 min three times at room temperature
Incubate the sections with secondary antibodies and DAPI for 1 hour at room temperature
Wash in PBS for 30 min three times at room temperature
Mount your slides using relevant mounting media and cover slip
Imaging
Imaging
Image the sections using a confocal microscope
Analysis
Analysis
Analyze the picture using ImageJ/FIJI