Immunohistochemistry Protocol for Free-floating Fixed Tissue

Wash sections (6 x 0h 10m 0s) in Dilution Media (DM) (0.2Molarity (M)TBS plus 0.05% volumeTriton X-100).

Wash sections for 0h 10m 0s in DM (1/6).

Wash sections for 0h 10m 0s in DM (2/6).

Wash sections for 0h 10m 0s in DM (3/6).

Wash sections for 0h 10m 0s in DM (4/6).

Wash sections for 0h 10m 0s in DM (5/6).

Wash sections for 0h 10m 0s in DM (6/6).

Endogenous peroxidase inhibition (0h 20m 0s). 0.1Molarity (M) Sodium meta-periodate in TBS.

• 100mL 0.2Molarity (M)Tris-buffered saline (TBS)
• 2.13g Sodium meta-periodate

Wash (2 x 0h 10m 0s) in DM.

Wash for 0h 10m 0s in DM (1/2).

Wash for 0h 10m 0s in DM (2/2).

Serum blocking step (1h 0m 0s incubation):

• 100mL DM
• 3mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 2g Bovine Serum Albumin (BSA)

Incubation in primary antibody (18h 0m 0s- 72h 0m 0s). See antibody catalog for concentration of primary antibody.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA
• 0.5mL Triton X-100
  Note

  Optionally, refrigerate 4°Cto keep antibody stable

Wash (6 x 0h 10m 0s) in DM.

Wash in DM for 0h 10m 0s (1/6).

Wash in DM for 0h 10m 0s (2/6).

Wash in DM for 0h 10m 0s (3/6).

Wash in DM for 0h 10m 0s (4/6).

Wash in DM for 0h 10m 0s (5/6).

Wash in DM for 0h 10m 0s (6/6).

Secondary antibody incubation (1h 0m 0s) Concentration of secondary antibody is always 1:200 in solvent.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA

Wash (6 x 0h 10m 0s) in DM.

Wash for 0h 10m 0s in DM (1/6).

Wash for 0h 10m 0s in DM (2/6).

Wash for 0h 10m 0s in DM (3/6).

Wash for 0h 10m 0s in DM (4/6).

Wash for 0h 10m 0s in DM (5/6).

Wash for 0h 10m 0s in DM (6/6).

Avidin-Biotin Complex (ABC) Step (1h 0m 0s) - Vectastain Elite ABC-HRP Kit (PK-6100).

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA

Add ABC Reagent A and B to 1/10th of total desired volume of solvent.

Incubate for 0h 30m 0s at Room temperature. Then dilute 1:10 using the same solvent.

A	B	C	D
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

Wash for 0h 10m 0s in DM.

Wash for 0h 10m 0s with TBS.

Wash (3 x 0h 10m 0s) in 0.2Molarity (M) Imidazole/1.0Molarity (M) Sodium Acetate buffer 7.2 to 7.4 .

• 1000mL dH₂O
• 0.68g Imidazole
• 6.8g Sodium Acetate.
• Retain 100mL of non-pH’d buffer for DAB preparation.

Wash for 0h 10m 0s in Imidazole/Sodium Acetate buffer (1/3).

Wash for 0h 10m 0s in Imidazole/Sodium Acetate buffer (2/3).

Wash for 0h 10m 0s in Imidazole/Sodium Acetate buffer (3/3).

DAB Step (Neutralize DAB with bleach when done)

Make DAB solution

• 100mL non-pH'd imidazole acetate buffer from above
• 50mg 3,3-Diaminobenzidine Tetrahydrochloride (DAB)
• 2g 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)

• 3mL of dH₂O
• 100µL of 30% (v/v) hydrogen peroxide (H₂O₂)

Start reaction -- add 500µL of 1% (v/v) hydrogen peroxide (H₂O₂) to the above DAB mixture just prior to use.

OR add 16.7µL of 30% (v/v) hydrogen peroxide (H₂O₂), per 100mL.

Place tissue in DAB solution.

• Develop tissue for approximately 0h 4m 0s to 0h 7m 0s.
• Timing is critical, ensure all tissue spends the same amount of time in DAB solution.

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 0h 10m 0s).

Wash developed tissue in imidazole acetate buffer for 0h 10m 0s (1/3).

Wash developed tissue in imidazole acetate buffer for 0h 10m 0s (2/3).

Wash developed tissue in imidazole acetate buffer for 0h 10m 0s (3/3).

Store tissue in 0.2Molarity (M) Phosphate-buffered saline (PBS) in refrigerator 4°C until mounted on slides.