Dec 01, 2023

Public workspaceImmunohistochemistry Protocol for Free-floating Fixed Tissue

DOI
dx.doi.org/10.17504/protocols.io.8epv5x3mdg1b/v1
  • 1Arizona State University
Scott Muller
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x3mdg1b/v1
Protocol Citation: Yaping Chu, Scott Muller, Jeffrey Kordower 2023. Immunohistochemistry Protocol for Free-floating Fixed Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x3mdg1b/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: October 19, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 89629
Keywords: Immunohistochemistry, ASAPCRN
Abstract
Immunohistochemistry protocol for staining free-floating fixed tissue in the Kordower Laboratory.
Attachments
nv29biaqf.docx
nv29biaqf.docx
17KB
Guidelines
HISTO- NOTES:
  • Primate tissue staining dishes use Amount100 mL solution per dish
  • Rodent tissue staining dishes Amount50 mL solution per dish
  • If staining a large number of primate cases, incubate 1' & 2' Ab in individual cups to conserve volume of Ab used.
  • Prepare bleach neutralizing solution prior to Step 12.
  • Be conscious of tissue saturation while washing and incubating. i.e. Check that tissue is fully submerged in solution & not clumping. This will ensure proper penetration of antibodies & other reagents.
  • Always include Positive & Negative Controls.
  • Positive: Use relevant control tissue to confirm specific antibody detection. (i.e. pS129; control tissue should consist of nigral sections previously successfully stained for pS129).
  • Negative: Ideally, use tissue that you know does not contain the targeted antigen. If not available, use a section of tissue not incubated in the 1' Ab (primary delete).
  • When incubating 1' Ab overnight, leave on shaker in refrigerator.
  • Can incubate in fridge on a shaker, covered in parafilm, over the weekend or up to 3 days.
  • Select a secondary antibody directed against the species in which the primary antibody was raised (i.e. if a primary antibody raised in rabbit is used, an anti-rabbit secondary antibody raised in a species other than rabbit must be used).
Materials
  • Dilution Media (DM) (Concentration0.2 Molarity (M) TBS plus Concentration0.05 % volume Triton X-100)
  • Concentration0.2 Molarity (M) Tris-buffered saline (TBS)
  • Sodium meta-periodate
  • Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • Bovine Serum Albumin (BSA)
  • Triton X-100
  • Vectastain Elite ABC-HRP Kit (PK-6100)
  • Imidazole
  • Sodium Acetate
  • 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
  • Concentration30 % (v/v) hydrogen peroxide
  • Concentration0.2 Molarity (M) Phosphate-buffered saline (PBS)
  • Household Bleach
  • Primary antibody against the target antigen
  • Secondary antibody directed against the species in which the primary antibody was raised (i.e. if a primary antibody raised in rabbit is used, an anti-rabbit secondary antibody raised in a species other than rabbit must be used).
DAY 1 (4 hrs)
DAY 1 (4 hrs)
Wash sections (6 x Duration00:10:00 ) in Dilution Media (DM) (Concentration0.2 Molarity (M) TBS plus Concentration0.05 % volume Triton X-100).
10m
Wash
Wash sections for Duration00:10:00 in DM (1/6).
10m
Wash sections for Duration00:10:00 in DM (2/6).
10m
Wash sections for Duration00:10:00 in DM (3/6).
10m
Wash sections for Duration00:10:00 in DM (4/6).
10m
Wash sections for Duration00:10:00 in DM (5/6).
10m
Wash sections for Duration00:10:00 in DM (6/6).
10m
Endogenous peroxidase inhibition (Duration00:20:00 ). Concentration0.1 Molarity (M) Sodium meta-periodate in TBS.
  • Amount100 mL Concentration0.2 Molarity (M) Tris-buffered saline (TBS)
  • Amount2.13 g Sodium meta-periodate
20m
Wash (2 x Duration00:10:00 ) in DM.
10m
Wash
Wash for Duration00:10:00 in DM (1/2).
10m
Wash for Duration00:10:00 in DM (2/2).
10m
Serum blocking step (Duration01:00:00 incubation):
  • Amount100 mL DM
  • Amount3 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • Amount2 g Bovine Serum Albumin (BSA)
1h
Incubation
Incubation in primary antibody (Duration18:00:00 - Duration72:00:00 ). See antibody catalog for concentration of primary antibody.
  • Amount100 mL DM
  • Amount1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • Amount1 g BSA
  • Amount0.5 mL Triton X-100
Note
**Optionally, refrigerate Temperature4 °C to keep antibody stable**

3d 18h
Incubation
DAY 2 (8 hrs)
DAY 2 (8 hrs)
2h 30m
Wash (6 x Duration00:10:00 ) in DM.
10m
Wash
Wash in DM for Duration00:10:00 (1/6).
10m
Wash in DM for Duration00:10:00 (2/6).
10m
Wash in DM for Duration00:10:00 (3/6).
10m
Wash in DM for Duration00:10:00 (4/6).
10m
Wash in DM for Duration00:10:00 (5/6).
10m
Wash in DM for Duration00:10:00 (6/6).
10m
Secondary antibody incubation (Duration01:00:00 ) Concentration of secondary antibody is always 1:200 in solvent.
  • Amount100 mL DM
  • Amount1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • Amount1 g BSA
1h
Incubation
Wash (6 x Duration00:10:00 ) in DM.
Note
**(incubate ABC in solvent during these washes)**.

10m
Incubation
Wash
Wash for Duration00:10:00 in DM (1/6).
10m
Wash for Duration00:10:00 in DM (2/6).
10m
Wash for Duration00:10:00 in DM (3/6).
10m
Wash for Duration00:10:00 in DM (4/6).
10m
Wash for Duration00:10:00 in DM (5/6).
10m
Wash for Duration00:10:00 in DM (6/6).
10m
Avidin-Biotin Complex (ABC) Step (Duration01:00:00 ) - Vectastain Elite ABC-HRP Kit (PK-6100).
  • Amount100 mL DM
  • Amount1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • Amount1 g BSA
1h
Add ABC Reagent A and B to 1/10th of total desired volume of solvent.
Pipetting
Incubate for Duration00:30:00 at TemperatureRoom temperature . Then dilute 1:10 using the same solvent.
Note
This is your working solution. See chart below for example volumes.
ABCD
Working Solution A (drops) B (drops) 1/10th Working solution
25 mL 1 1 2.5 mL
50 mL 2 2 5 mL
100 mL 4 4 10 mL

30m
Incubation
Wash for Duration00:10:00 in DM.
10m
Wash
Wash for Duration00:10:00 with TBS.
10m
Wash
Wash (3 x Duration00:10:00 ) in Concentration0.2 Molarity (M) Imidazole/Concentration1.0 Molarity (M) Sodium Acetate buffer Ph7.2 to Ph7.4 .
  • Amount1000 mL dH2O
  • Amount0.68 g Imidazole
  • Amount6.8 g Sodium Acetate.
  • Retain Amount100 mL of non-pH’d buffer for DAB preparation.
10m
Wash
Wash for Duration00:10:00 in Imidazole/Sodium Acetate buffer (1/3).
10m
Wash for Duration00:10:00 in Imidazole/Sodium Acetate buffer (2/3).

10m
Wash for Duration00:10:00 in Imidazole/Sodium Acetate buffer (3/3).
10m
DAB Step (Neutralize DAB with bleach when done)
Imaging
Make DAB solution

  • Amount100 mL non-pH'd imidazole acetate buffer from above
  • Amount50 mg 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
  • Amount2 g Nickel(II) sulfate hexahydrate **(Only used with certain primary antibodies, chromagen enhancer that changes brown DAB precipitate to blue-purple)**
Make Concentration1 % (v/v) Hydrogen Peroxide (H2O2)

  • Amount3 mL of dH2O
  • Amount100 µL of Concentration30 % (v/v) hydrogen peroxide (H2O2)
Start reaction -- add Amount500 µL of Concentration1 % (v/v) hydrogen peroxide (H2O2) to the above DAB mixture just prior to use.
OR add Amount16.7 µL of Concentration30 % (v/v) hydrogen peroxide (H2O2), per Amount100 mL .
Place tissue in DAB solution.
  • Develop tissue for approximately Duration00:04:00 to Duration00:07:00 .
  • Timing is critical, ensure all tissue spends the same amount of time in DAB solution.
11m
To monitor signal, move all tissue to imidazole buffer, remove one section and mount on an UNSUBBED slide and view under microscope. Place all tissue back in DAB solution to increase signal intensity, if needed.
Wash developed tissue in imidazole acetate buffer (3 x Duration00:10:00 ).

Note
**Neutralize DAB with BLEACH!!!**

10m
Wash
Wash developed tissue in imidazole acetate buffer for Duration00:10:00 (1/3).
10m
Wash developed tissue in imidazole acetate buffer for Duration00:10:00 (2/3).
10m
Wash developed tissue in imidazole acetate buffer for Duration00:10:00 (3/3).
10m
Store tissue in Concentration0.2 Molarity (M) Phosphate-buffered saline (PBS) in refrigerator Temperature4 °C until mounted on slides.