Oct 20, 2023

Public workspaceImmunohistochemistry Protocol for Free-floating Fixed Tissue with Tyramine Signal Amplification (TSA)

DOI
dx.doi.org/10.17504/protocols.io.j8nlkom2dv5r/v1
  • Yaping Chu1,
  • Jeremy Molina1,
  • Scott Muller1,
  • Jeffrey H Kordower1
  • 1Arizona State University
  • Team Kordower
Scott Muller
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkom2dv5r/v1
Protocol Citation: Yaping Chu, Jeremy Molina, Scott Muller, Jeffrey H Kordower 2023. Immunohistochemistry Protocol for Free-floating Fixed Tissue with Tyramine Signal Amplification (TSA). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkom2dv5r/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: May 31, 2024
Protocol Integer ID: 89625
Keywords: Immunohistochemistry, ASAPCRN
Abstract
Immunohistochemistry protocol for staining free-floating fixed tissue with Tyramine signal amplification (TSA) in the Kordower Laboratory. TSA allows a much lower concentration of primary antibody to be used (typically 1:10K or 1:15K) at the expense of an extra day of staining.
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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
  • Sodium tetraborate decahydrate
  • Boric Acid
  • Biotin tyramide
  • 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
  • Nickel(II) sulfate hexahydrate
  • 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 (3.5hrs)
DAY 1 (3.5hrs)
Wash sections (6 x Duration00:05:00 ) in Dilution Media (DM) (Concentration0.2 Molarity (M) TBS plus Concentration0.05 % volume Triton X-100).
5m
Wash
Wash sections for Duration00:05:00 in DM (1/6).
5m
Wash sections for Duration00:05:00 in DM (2/6).
5m
Wash sections for Duration00:05:00 in DM (3/6).
5m
Wash sections for Duration00:05:00 in DM (4/6).
5m
Wash sections for Duration00:05:00 in DM (5/6).
5m
Wash sections for Duration00:05:00 in DM (6/6).
5m
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 Duration00:00:00 ) in DM.
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 (typically 1:10K or 1:15K with TSA).
  • 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 (4hrs):
DAY 2 (4hrs):
Wash (6 x Duration00:05:00 ) in DM.

5m
Wash
Wash for Duration00:05:00 in DM (1/6).

5m
Wash for Duration00:05:00 in DM (2/6).
5m
Wash for Duration00:05:00 in DM (3/6).
5m
Wash for Duration00:05:00 in DM (4/6).
5m
Wash for Duration00:05:00 in DM (5/6).
5m
Wash for Duration00:05:00 in DM (6/6).
5m
Incubation in secondary antibody (Duration01:00:00 ). Concentration of secondary antibody is always 1:500 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
Wash (6 x Duration00:05:00 ) in DM.
Note
**(incubate ABC in solvent during these washes)**

5m
Wash
Wash for Duration00:05:00 in DM (1/6).

5m
Wash for Duration00:05:00 in DM (2/6).
5m
Wash for Duration00:05:00 in DM (3/6).
5m
Wash for Duration00:05:00 in DM (4/6).
5m
Wash for Duration00:05:00 in DM (5/6).
5m
Wash for Duration00:05:00 in DM (6/6).
5m
Actin Biotin Complex Step (Duration01:00:00 ) - Vectastain Elite ABC-HRP Kit (PK-6100).

Note
**Re-use for step 15. Can be stored overnight in refrigerator Temperature4 °C **
  • 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.
Mix
Incubate for Duration00:30:00 at TemperatureRoom temperature .
Note
Then dilute 1:10 using the same solvent. 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 2.5mL
50 mL 2 2 5mL
100mL 4 4 10mL

30m
Incubation
Wash for Duration00:10:00 in DM.

10m
Wash
Wash Duration00:10:00 in TBS.

10m
Wash
Wash (2 x Duration00:10:00 ) in Concentration0.05 Molarity (M) Borate buffer Ph8.5 .
  • Amount1000 mL dH2O
  • Amount4.77 g Sodium tetraborate decahydrate
  • Amount2.32 g Boric Acid
10m
Wash
Wash for Duration00:10:00 in Borate buffer (1/2).

10m
Wash for Duration00:10:00 in Borate buffer (2/2).

10m
Incubate sections with biotin tyramide solution (Duration00:30:00 ).
Note
DO NOT USE IF >6 MONTHS OLD.
  • Amount100 mL Borate buffer
  • Amount2 µL of Concentration50 mg/mL biotin tyramide stock
  • Amount10 µL Concentration30 % (v/v) Hydrogen Peroxide (H2O2)
30m
Incubation
Wash (3 x Duration00:10:00 ) in TBS (Antigen is now labeled with biotin).
10m
Wash
Wash for Duration00:10:00 in TBS (1/3).
10m
Wash for Duration00:10:00 in TBS (2/3).
10m
Wash for Duration00:10:00 in TBS (3/3).
10m
DAY 3 (4 hrs):
DAY 3 (4 hrs):
40m
40m
Repeat Actin Biotin Complex Step 9 then 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)
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 .

Pipetting
Place tissue in DAB solution.
  • Develop tissue for approximately Duration00:05:00 .
  • Timing is critical, ensure all tissue spends the same amount of time in DAB solution.
5m
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.
Imaging
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.