3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections

madalynn.erb Erb

Apr 12, 2024

3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections

DOI

https://dx.doi.org/10.17504/protocols.io.3byl497zjgo5/v1

madalynn.erb Erb¹

¹Van Andel Research Institute

Team Lee

Jane Balster

ASAP - Team Lee

DOI: https://dx.doi.org/10.17504/protocols.io.3byl497zjgo5/v1

Protocol Citation: madalynn.erb Erb 2024. 3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl497zjgo5/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 08, 2024

Last Modified: May 31, 2024

Protocol Integer ID: 98141

Keywords: ASAPCRN, immunohistochemistry on mouse brain tissue section, floating mouse brain section, mouse brain tissue section, mouse brain section, mouse brain, diaminobenzidine tetrahydrochloride, dab, immunohistochemistry, mouse

Abstract

This protocol details the staining for free floating mouse brain sections.

Materials

 Pyrex® Staining DishTed Pella Inc.Catalog #36754-60 
 8-Section Staining NetsTed Pella Inc.Catalog #36154-64 
VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard)Vector LaboratoriesCatalog #PK-6100 
Pierce™ DAB Substrate KitThermo ScientificCatalog #34002  
Fisherbrand™ Superfrost™ Plus Microscope SlidesFisher ScientificCatalog #12-550-15  
EntellanElectron Microscopy SciencesCatalog #14802  

Recipes

10X TBS

AB
1M Tris500 ml
5M NaCl300 ml 
H2O200 mL 

1X TBS + 0.1% triton → 1 liter

AB
10X TBS100 ml
Triton X1 ml
Water899 ml 

TBS → 1 liter

AB
1M Tris50 ml
5M NaCl30ml
Water919 ml
 
TBST (0.1% Tween-20) → 1 liter

AB
10X TBS100 ml
Tween-201ml
Water899 ml
 
0.2M Phosphate Buffer
 
AB
Na2HPO422.72 g 
NaH2PO45.52 g 
H2O1000 mL 

 0.1M Phosphate Buffer

AB
0.2M PB500 mL 
H2O500 mL 
 
Cresyl Violet (500 mL)
 
AB
H2O500 mL 
Cresyl Violet Acetate0.5g 
100% Glacial Acetic Acid1.25mL
Warm to 60°C and stir until completely dissolved filter with whatman paper  (wrap in foil to protect from light)
 
Quenching solution→100ml
 
AB
30% hydrogen peroxide (Sigma H1009)1 ml
Methanol99 ml
 
ABC mix→60ml
 
AB
TBS60 ml
Reagent A24 drops
Reagent B24 drops
 
DAB Solution→30ml
 
AB
PBS30 ml
(DAB reagent 1)12 drops buffer pH = 7.5
(DAB reagent 2)24 drops DAB
(DAB reagent 3)12 drops hydrogen peroxide
vortex between reagents
 
Blocking solution→100ml
 
AB
NGS10 ml
TBS + triton90 ml + 0.1%
 
 Primary Antibodies:
ABCD
 Target  Species  Conc  Manufacturer 
 Iba1  Rabbit  1:1000  WAKO 019-19741 
 GFAP  Mouse  1:1000  Sigma G3893 
 pSer129-Synuclein  Rabbit  1:1000  Abcam ab51253 
 TH  Rabbit  1:2000  Novus Biological N300109 
 pSer202, pThr205 Tau (AT8)  Mouse  1:1000  Thermofisher MN1020 
 GFP  Rabbit  1:1000  Thermofisher A-11122 
 Secondary Antibodies: 
ABCDE
 Target  Species  Conc  Manufacturer    
 Mouse  Goat  1:500  BA-9200-1.5  Biotinylated 
 Rabbit  Goat  1:500  BA-1000-1.5  Biotinylated 
 

Day 1

2d 1h 35m

Staining protocol for 35μm free floating mouse brain sections.

Staining is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64) .

The sections can be transferred between wells using a paint brush.

Volume of solution:

20 mL  -30 mL   for antibodies or ABC mix .
50 mL   for washing.

Gently rock the plates during the washing and incubation steps.

Wash sections: 3 times (5 min per wash) in Tris Buffer Saline (TBS) at Room temperature  .

Wash sections: Wash for 00:05:00   in Tris Buffer Saline (TBS) at Room temperature  (1/3).

Wash sections: Wash for 00:05:00   in Tris Buffer Saline (TBS) at Room temperature  (2/3).

Wash sections: Wash for 00:05:00   in Tris Buffer Saline (TBS) at Room temperature  (3/3).

Quenching: Incubate sections in Quenching Solution (0.3% H202 in Methanol) for 00:10:00   at 4 °C   .

10m

Wash sections: 3 short rinses in TBS then 2 times 5min in TBS at Room temperature  .

Then wash for 00:05:00   in TBS at Room temperature  (1/2).

Then wash for 00:05:00   in TBS at Room temperature  (2/2).

Block sections in blocking solution (10% Normal Goat Serum (NGS) + 0.1% Triton-X in TBS) for 01:00:00   at Room temperature  .

Primary Antibodies: Dilute antibodies in 5% NGS + 0.1% Triton-X in TBS.

Incubate in primary antibody solution for 48:00:00   at 4 °C  .

Cover dishes with parafilm for this step.

Day 4

1d 3h 47m

Wash 3 times (10 min each wash) in TBST (0.1% Tween 20 in TBS) at Room temperature  .

Wash for 00:10:00  in TBST (0.1%Tween 20 in TBS) at Room temperature  (1/3).

10m

Wash for 00:10:00  in TBST (0.1%Tween 20 in TBS) at Room temperature  (2/3).

10m

Wash for 00:10:00  in TBST (0.1%Tween 20 in TBS) at Room temperature  (3/3).

10m

Secondary Antibodies: 

Dilute secondary antibodies in TBST.

Biotinylated goat anti-rabbit IgG (1:500 dilution) or Biotinylated goat anti-mouse IgG (1:500 dilution).

Incubate for 02:00:00   atRoom temperature   or 24:00:00   at 4 °C  .

1d 2h

Wash sections 3 times (10min each wash) in TBST at Room temperature  .

Wash for 00:10:00  in TBST at Room temperature  (1/3).

10m

Wash for 00:10:00  in TBST at Room temperature  (2/3).

10m

Wash for 00:10:00  in TBST at Room temperature  (3/3).

10m

Prepare ABC solution using VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) (PK-6100).

ABC mix: 5 mL   TBS + 2 drops of reagent A (vortex) + 2 drops reagent B (vortex) let stand at Room temperature   for 00:30:00   before using.

30m

Vortex between reagents (after adding reagent A and after adding reagent B).

Make 30 mL   ABC solution per dish.

Incubate sections in ABC solution for 01:00:00   at Room temperature  .

Wash sections 3 times (10min each wash) in TBST at Room temperature  .

Wash for 00:10:00  in TBST at Room temperature  (1/3).

10m

Wash for 00:10:00  in TBST at Room temperature  (2/3).

10m

Wash for 00:10:00  in TBST at Room temperature  (3/3).

10m

Wash sections 2 times (5 min each wash) in TBS at Room temperature  .

Wash sections for 00:05:00   in TBS at Room temperature  (1/2).

Wash sections for 00:05:00   in TBS at Room temperature  (2/2).

Prepare DAB solution using DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine) (SK-4100).

5 mL   TBS + 2 drops of Reagent 1 (Buffer pH=7.5) + 4 drops Reagent 2 (DAB) + 2 drops Reagent 3 (H2O2).
Vortex between reagents (after reagent 1, after reagent 2 and after reagent 3).
Make 30 mL   DAB solution per dish.

Revelation with DAB chromogen (until cells are visible):

Note
typically about 00:02:00   for TH.
All dishes must be developed for the same amount of time – do them together, ask a friend for help if you have more than 2 dishes of tissue.

Perform 3 short rinses in TBS at Room temperature  .

Perform rinse in TBS at Room temperature  (1/3).

Perform rinse in TBS at Room temperature  (2/3).

Perform rinse in TBS at Room temperature  (3/3).

Wash sections 5 time (5 min each wash) in TBS at Room temperature  .

Wash sections for 00:05:00   in TBS at Room temperature  (1/5).

Wash sections for 00:05:00   in TBS at Room temperature  (2/5).

Wash sections for 00:05:00   in TBS at Room temperature  (3/5).

Wash sections for 00:05:00   in TBS at Room temperature  (4/5).

Wash sections for 00:05:00   in TBS at Room temperature  (5/5).

Mount sections:

Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).

This is easiest to do using a medium sized paint brush.

After mounting let slides dry at Room temperature   (00:02:00  -00:10:00  ) then dip in miliQ H2O.

12m

Dry slides Overnight   at Room temperature   before moving to the next step.

Day 5 or 6

9h 18m

Rehydrate slides in H2O for 00:02:00  -00:03:00   at Room temperature  .

Dehydrate slides in sequential EtOH washes (70%, 95%, 100% EtOH) for 00:05:00   each wash at Room temperature  .

Clear tissue for 00:05:00   in fresh Xylene I then incubate in fresh Xylene for ≥ 00:05:00  .

a.	Keep slides in Xylene until coversliping.
b.	Perform Xylene steps and coversliping in a chemical fume hood.

10m

Use Entellan mounting medium (Electron Microscopy Sciences 14802) to coverslip slides.

Apply one line of Entellan across the middle of the slide.

Cover with glass coverslip and gently press down with forceps.

Let slides dry in the fume hood for 01:00:00   – then dry on the bench Overnight   at Room temperature  .

1h 5m

Gently remove excess Entellen with a razor blade.

Optional Cresyl Violet Protocol (NISSL stain)

10m

After rehydration and before ethanol steps (dehydration).

Incubate slides in fresh cresyl violet for 00:10:00   at Room temperature  .

10m

Proceed with dehydration steps normally.

Must stain with cresyl violet for TH stereology in substantia nigra.

Secondary Antibodies

 
ABCDE
 Target  Species  Conc  Manufacturer    
 Mouse  Goat  1:500  BA-9200-1.5  Biotinylated 
 Rabbit  Goat  1:500  BA-1000-1.5  Biotinylated 
 

	A	B
	H2O	500 mL
	Cresyl Violet Acetate	0.5g
	100% Glacial Acetic Acid	1.25mL

	A	B
	PBS	30 ml
	(DAB reagent 1)	12 drops buffer pH = 7.5
	(DAB reagent 2)	24 drops DAB
	(DAB reagent 3)	12 drops hydrogen peroxide

A	B	C	D
Target	Species	Conc	Manufacturer
Iba1	Rabbit	1:1000	WAKO 019-19741
GFAP	Mouse	1:1000	Sigma G3893
pSer129-Synuclein	Rabbit	1:1000	Abcam ab51253
TH	Rabbit	1:2000	Novus Biological N300109
pSer202, pThr205 Tau (AT8)	Mouse	1:1000	Thermofisher MN1020
GFP	Rabbit	1:1000	Thermofisher A-11122

	A	B
	1M Tris	500 ml
	5M NaCl	300 ml
	H2O	200 mL

	A	B
	10X TBS	100 ml
	Triton X	1 ml
	Water	899 ml

	A	B
	1M Tris	50 ml
	5M NaCl	30ml
	Water	919 ml

	A	B
	10X TBS	100 ml
	Tween-20	1ml
	Water	899 ml