Nov 15, 2019

Public workspaceBDA Histology

DOI
dx.doi.org/10.17504/protocols.io.z4af8se
  • 1University of Western Ontario
Blake Butler
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DOI: dx.doi.org/10.17504/protocols.io.z4af8se
Protocol Citation: Blake Butler 2019. BDA Histology. protocols.io https://dx.doi.org/10.17504/protocols.io.z4af8se
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 16, 2019
Last Modified: November 15, 2019
Protocol Integer ID: 22370
Abstract
Staining protocol for visualization of BDA 3K.
Species - Feline
Tissue Preparation - Perfusion of 4% PAF; Cryoprotected 30% Sucrose; Section thickness of 60 microns
Materials
STEP MATERIALS
Reagent3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
ReagentAvidin/Biotin HRP ComplexVector LaboratoriesCatalog #PK-6100
ReagentNormal Goat SerumCedarlaneCatalog #CL1200-500
Protocol materials
Reagent3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
ReagentAvidin/Biotin HRP ComplexVector LaboratoriesCatalog #PK-6100
ReagentNormal Goat SerumCedarlaneCatalog #CL1200-500
ReagentAvidin/Biotin HRP ComplexVector LaboratoriesCatalog #PK-6100
ReagentNormal Goat SerumCedarlaneCatalog #CL1200-500
Reagent3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
Preparatory Steps
Preparatory Steps
Reconstitute the Vectastain ABC solution

Prepare 80 mL solution:
Amount79 mL Concentration0.1 Molarity (M) Physiological Buffer
Amount480 µL Solution A
Amount480 µL Solution B
ReagentAvidin/Biotin HRP ComplexVector LaboratoriesCatalog #PK-6100

Thaw 6 x 1mL vials of NGS for later use
ReagentNormal Goat SerumCedarlaneCatalog #CL1200-500



Histological Reaction
Histological Reaction


Rinse sections in 0.1 M Physiological Buffer (4x5min)

Duration00:05:00
Duration00:05:00
Duration00:05:00
Duration00:05:00
Block Endogenous Peroxidase

Amount105 mL Methanol
Amount45 mL De-ionized Water
Amount750 µL Concentration30 % volume Hydrogen Peroxide

Duration00:30:00

Rinse sections in 0.1 M Physiological Buffer (4x5min)

Duration00:05:00
Duration00:05:00
Duration00:05:00
Duration00:05:00

Transfer tissue to individual reaction wells and incubate in Avidin/Biotin HRP Complex Prepared in Step 1

Duration01:30:00
Transfer tissue back to reaction trays and rinse sections in 0.1 M Physiological Buffer (3x10min)

Duration00:10:00
Duration00:10:00
Duration00:10:00

Incubate in DAB-Nickel Chromogen Solution


Amount150 mL Concentration0.1 Molarity (M) Physiological Buffer
Amount.1125 g 3,3' Diaminobenzidine Tetrachloride (DAB)
Amount1.875 mL Concentration1 Mass / % volume Cobalt Chloride (Added dropwise)
Amount3 mL Concentration1 Mass / % volume Nickel Ammonium Sulfate (Added dropwise)
Amount50 µL Concentration30 % volume Hydrogen Peroxide (Added 60s before incubation)

Reagent3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
Duration00:20:00 (Approximate time - Actual endpoint determined visually)

Rinse sections in 0.1 M Physiological Buffer (3x5min)

Duration00:05:00
Duration00:05:00
Duration00:05:00
Mount from 0.01 M Physiological Buffer