May 25, 2017
Version 2
NeuN Immunohistochemistry Protocol V.2
- Nara Gyzely de Morais Magalhães 1,
- Cristovam Guerreiro Diniz and Cristovam Wanderley Picanço Diniz1
- 1Universidade Federal do Pará - Instituto Federal do Pará
Protocol Citation: Nara Gyzely de Morais Magalhães , Cristovam Guerreiro Diniz and Cristovam Wanderley Picanço Diniz 2017. NeuN Immunohistochemistry Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.h56b89e
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
Created: May 24, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 6046
Keywords: IHC
Abstract
Protocol for Immunohistochemistry free-floating sections with anti-NeuN antibody for avian tissue.
Materials
MATERIALS
Boric acidP212121
Ammonium ChlorideP212121
Sodium Acetate, Trihydrate
Sodium Phosphate monobasic
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3264
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100
Ammonium nickel(II) sulfate hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #574988
3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
α-D-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #158968
Normal Goat Serum Blocking SolutionVector LaboratoriesCatalog #S-1000
Anti-NeuN Antibody, clone A60Merck Millipore (EMD Millipore)Catalog #MAB377
Biotinylated Goat Anti-Mouse IgG AntibodyVector LaboratoriesCatalog #BA-9200
PBS
Antigen Retrieval Method
Antigen Retrieval Method
To remove the excess paraformaldehyde fixative, wash with 0,1 M PBS the sections free-floating at agitation in room temperature for 3 min. (1/3)
To remove the excess paraformaldehyde fixative, wash with 0,1 M PBS the sections free-floating at agitation in room temperature for 3 min. (2/3)
To remove the excess paraformaldehyde fixative, wash with 0,1 M PBS the sections free-floating at agitation in room temperature for 3 min. (3/3)
Incubate with 12% Boric acid (pH =9,0, 70°C) in a water bath for 1 hour.
Note
When the temperature reaches 50°C, using a brush put the sections at the recipient and wait the temperature reaches 70°C and keep at this temperature for 1 hour.
Remove the recipients from water bath and wait until it is at room temperature.
Wash the sections with 0,1% PBS/T for 5 minutes. (1/3)
Wash the sections with 0,1% PBS/T for 5 minutes. (2/3)
Wash the sections with 0,1% PBS/T for 5 minutes. (3/3)
Wash the sections with 0,1M PBS for 2 minutes (shaking). (1/3)
Wash the sections with 0,1M PBS for 2 minutes (shaking). (2/3)
Wash the sections with 0,1M PBS for 2 minutes (shaking). (3/3)
Protein Blocking Step
Protein Blocking Step
Incubate with blocking buffer (Normal Goat Serum Blocking Solution S-1000 10% in 0,3% PBST ) for 12 hours at gentle agitation in refrigeration 4°C.
Primary Antibody
Primary Antibody
Remove the serum and incubate with Anti NeuN Antibody (MAB377 Anti-NeuN Antibody, clone A60), diluted in 0,3%PBS/T at gentle agitation, overnight at 4°C.
Wash the sections with 0,1M PBS/T 0,1% for 2 minutes (shaking). (1/3)
Wash the sections with 0,1M PBS/T 0,1% for 2 minutes (shaking). (2/3)
Wash the sections with 0,1M PBS/T 0,1% for 2 minutes (shaking). (3/3)
Secondary Antibody
Secondary Antibody
Incubate with secondary antibody (Biotinylated Goat Anti-Mouse IgG Antibody, BA-9200, Vector Laboratories ) diluted in 0,3% PBS/T at 1:250, for 1 hour at room temperature.
Blocking Step
Blocking Step
Incubate with 0,3% hydrogen peroxide (diluted in 0,1 M PBS) for 15 minutes with light shaking.
Wash sections with PBS/T 0,1% for 2 minutes (shaking).(1/3)
Wash sections with PBS/T 0,1% for 2 minutes (shaking).(2/3)
Wash sections with PBS/T 0,1% for 2 minutes (shaking).(3/3)
ABC
ABC
Incubate in VECTASTAIN® ABC KIT solution for 1 hour (total) at 4ºC with light shaking.
First 37,5 µl A + 37,5µl B with 1,88 ml 0,3%PBS/T for 30 minutes.
Add 13,12 ml 0,3%PBS/T and incubate for 30 more minutes at 4ºC with light shaking.
Wash with 0,1% PBS /T for 5 minutes, (shaking). (1/2)
Wash with 0,1% PBS /T for 5 minutes, (shaking). (2/2)
DAB Visualization-GDN preparation
DAB Visualization-GDN preparation
Firstly prepare the Solution A by mix 0,006g of Diaminobenzidine (DAB) with 5 ml of distilled water.
Secondly, prepare the Solution B by mixing 0,250g of Nickel ammonium sulfate with 5 ml de Acetate Buffer pH 6.0.
Thirdly mix Solution A and B adding ammonium chloride (0,004g) with 0.020g α-D-Glucose.
Leave the section in this mix of A and B for 5 minutes.
DAB Visualization
DAB Visualization
Incubate sections with solution GND and wait for 3 minutes.
Note
After each step remove the remaining solution from the previous step with a pipette, with careful not to damage or lose sections.
Use only sterilized material to minimize the risk of contamination between different antibodies or solutions.
At the end add chlorine to inactivate GND + DAB residual solution before disposal on an appropriate container.
Add 0,007g of Glucose-oxidase for each 3ml of GND solution for revelation. Stop revelation when the goal contrast is achieved (use a low gain microscope).
Remove the GDN + Glucose oxidase and wash the sections using 0,1M PBS for 2 minutes with light shaking. (1/3)
Remove the GDN + Glucose oxidase and wash the sections using 0,1M PBS for 2 minutes with light shaking. (2/3)
Remove the GDN + Glucose oxidase and wash the sections using 0,1M PBS for 2 minutes with light shaking. (3/3)
Mount the sections in appropriate gelatinized microscope slides and dry at room temperature for 12 hours or more depending on the mounting medium of choice.
Dihydrate and add the coverslips.