Oct 20, 2023

Immunohistochemistry Protocol for Free-floating Fixed Tissue

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzx725gx1/v1
  • 1Arizona State University
  • Team Kordower
Scott Muller
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzx725gx1/v1
Protocol Citation: Yaping Chu, Jeremy Molina, Scott Muller, Jeffrey Kordower 2023. Immunohistochemistry Protocol for Free-floating Fixed Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx725gx1/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 18, 2023
Last Modified: October 20, 2023
Protocol  Integer ID: 89626
Keywords: Immunohistochemistry, floating fixed tissue immunohistochemistry protocol, fixed tissue immunohistochemistry protocol, floating fixed tissue, tissue in the kordower laboratory, immunohistochemistry protocol, fixed tissue, tissue
Abstract
Immunohistochemistry protocol for staining free-floating fixed tissue in the Kordower Laboratory.
Attachments
Icon for nv29biaqf.docx
nv29biaqf.docx
17KB
Guidelines
HISTO- NOTES:
  • Primate tissue staining dishes use 100 mL solution per dish
  • Rodent tissue staining dishes 50 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) (0.2 Mass Percent TBS plus 0.05 % volume Triton X-100)
  • 0.2 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)
  • 30 % (v/v) hydrogen peroxide
  • 0.2 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)
Wash sections (6 x 00:10:00 ) in Dilution Media (DM) (0.2 Mass Percent TBS plus 0.05 % volume Triton X-100).
10m
Wash sections for 00:10:00 in DM (1/6).
10m
Wash sections for 00:10:00 in DM (2/6).
10m
Wash sections for 00:10:00 in DM (3/6).
10m
Wash sections for 00:10:00 in DM (4/6).
10m
Wash sections for 00:10:00 in DM (5/6).
10m
Wash sections for 00:10:00 in DM (6/6).
10m
Endogenous peroxidase inhibition (00:20:00 ). 0.1 Mass Percent Sodium meta-periodate in TBS.
  • 100 mL 0.2 Molarity (M) Tris-buffered saline (TBS)
  • 2.13 g Sodium meta-periodate
20m
Wash (2 x 00:10:00 ) in DM.
10m
Wash for 00:10:00 in DM (1/2).
10m
Wash for 00:10:00 in DM (2/2).
10m
Serum blocking step (01:00:00 incubation):
  • 100 mL DM
  • 3 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • 2 g Bovine Serum Albumin (BSA)
1h
Incubation in primary antibody (18:00:00 - 72:00:00 ). See antibody catalog for concentration of primary antibody.
  • 100 mL DM
  • 1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • 1 g BSA
  • 0.5 mL Triton X-100
Note
**Optionally, refrigerate 4 °C to keep antibody stable**

3d 18h
DAY 2 (8 hrs)
2h 30m
Wash (6 x 00:10:00 ) in DM.
10m
Wash in DM for 00:10:00 (1/6).
10m
Wash in DM for 00:10:00 (2/6).
10m
Wash in DM for 00:10:00 (3/6).
10m
Wash in DM for 00:10:00 (4/6).
10m
Wash in DM for 00:10:00 (5/6).
10m
Wash in DM for 00:10:00 (6/6).
10m
Secondary antibody incubation (01:00:00 ) Concentration of secondary antibody is always 1:200 in solvent.
  • 100 mL DM
  • 1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • 1 g BSA
1h
Wash (6 x 00:10:00 ) in DM.
Note
**(incubate ABC in solvent during these washes)**.

10m
Wash for 00:10:00 in DM (1/6).
10m
Wash for 00:10:00 in DM (2/6).
10m
Wash for 00:10:00 in DM (3/6).
10m
Wash for 00:10:00 in DM (4/6).
10m
Wash for 00:10:00 in DM (5/6).
10m
Wash for 00:10:00 in DM (6/6).
10m
Avidin-Biotin Complex (ABC) Step (01:00:00 ) - Vectastain Elite ABC-HRP Kit (PK-6100).
  • 100 mL DM
  • 1 mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
  • 1 g BSA
1h
Add ABC Reagent A and B to 1/10th of total desired volume of solvent.
Incubate for 00:30:00 at Room 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
Wash for 00:10:00 in DM.
10m
Wash for 00:10:00 with TBS.
10m
Wash (3 x 00:10:00 ) in 0.2 Mass Percent Imidazole/1.0 Mass Percent Sodium Acetate buffer 7.2 to 7.4 .
  • 1000 mL dH2O
  • 0.68 g Imidazole
  • 6.8 g Sodium Acetate.
  • Retain 100 mL of non-pH’d buffer for DAB preparation.
10m
Wash for 00:10:00 in Imidazole/Sodium Acetate buffer (1/3).
10m
Wash for 00:10:00 in Imidazole/Sodium Acetate buffer (2/3).

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

  • 100 mL non-pH'd imidazole acetate buffer from above
  • 50 mg 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
  • 2 g Nickel(II) sulfate hexahydrate **(Only used with certain primary antibodies, chromagen enhancer that changes brown DAB precipitate to blue-purple)**
Make 1 % (v/v) Hydrogen Peroxide (H2O2)

  • 3 mL of dH2O
  • 100 µL of 30 % (v/v) hydrogen peroxide (H2O2)
Start reaction -- add 500 µL of 1 % (v/v) hydrogen peroxide (H2O2) to the above DAB mixture just prior to use.
OR add 16.7 µL of 30 % (v/v) hydrogen peroxide (H2O2), per 100 mL .
Place tissue in DAB solution.
  • Develop tissue for approximately 00:04:00 to 00:07:00 .
  • 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 00:10:00 ).

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

10m
Wash developed tissue in imidazole acetate buffer for 00:10:00 (1/3).
10m
Wash developed tissue in imidazole acetate buffer for 00:10:00 (2/3).
10m
Wash developed tissue in imidazole acetate buffer for 00:10:00 (3/3).
10m
Store tissue in 0.2 Molarity (M) Phosphate-buffered saline (PBS) in refrigerator 4 °C until mounted on slides.