Immunohistochemistry protocols

Standard recipes and abbreviations

All sections sliced on a sliding vibratome (Model VT1200 S, Leica Biosystems, Concord, ON; with modifications by Peira Scientific Instruments, Belgium) at 50μm. Maintained in 1 x PBS until stained.

10xTBS
Trish-buffered saline (TBS; 0.5M)
TBS-TX recipe
10%, 10 x TBS +2.5% Triton X (20%)
10 x PBS
phosphate buffer saline
PBS-TX
PBS with Triton X (1 X PBS + 0.3% Triton X (100%))

Parvalbumin protocol

Day 1

  1. 3 x 10 min 1 x PBS washes
  2. Pre-Incubation/ blocking step for 1.5h on shaking rotator room temp.
    • 10% TBS(10x)
    • 2.5% Triton X (20%)
    • 5% Normal Goat serum
  3. 3 X 10 min TBS-TX washes
  4. Primary anti-body incubation for 48h at 4 degree Celsius on shaking rotator
    • Incubate in monoclonal primary mouse antibody, diluted at 1:2000 in TBS-TX (Sigma Aldrich, Monoclonal mouse anti-pervalbumin antibody, P3088, 0.2ml).

Day 2

  1. 3 X 10 min TBS-TX washes
  2. Secondary incubation for 90min at room temperature on shaking rotator
    • • Secondary goat anti mouse, diluted at 1:500 in TBS-TX (Sigma Aldrich, Biotinylated goat anti-mouse antibody, B7151, 1ml).
  3. 3 X 10 min TBS-TX washes
  4. 4) Vectastain ABC kit (Vector Laboratories, Vectastain ABC, PK-4000, 1 Kit)
    • Follow instruction for standard reaction
  5. 3 X 10 min TBS-TX washes
  6. DAB Reaction- (place wells on shaking rotator, approximately 5 min reaction. DAB staining takes place very quickly, start with a small amount of tissue to gauge time). When making DAB stock solution work quickly, and do not leave solution at room temperature. DAB oxidises quickly. New DAB stock solution will stain tissue faster than old solutions.
    • 5% DAB (Sigma Aldrich, 3,3′-Diaminobenzidine tetrahydrochloride hydrate D5637, 1g) stock solution
    • 10% 10 x TBS
    • 0.05% H2O2 (30%)
    • Nano pure H2O
  7. 3, 1 x PBS washes- (once desired staining has taken place immediately remove tissue from DAB solution and wash in 1 x PBS. Tissue will continue to darken in the first 2, 1 x PBS washes- Do no over stain). Wash for approximate 1 min in each 1 x PBS wash.
  8. Dehydrate in ethanol, clear in Hemo-De, cover slip with permount (Fisher Chemical Permount™ Mounting Medium, SP15-500).

Anterograde BDA tracer DAB (Ni2Cl intensified) reaction

Day 1

  1. 1, 10 min PBS wash
  2. 2 x 10 min PBS-TX washes
  3. 3 X 10 min TBS-TX washes
  4. Vectastain ABC kit (Vector Laboratories, Vectastain ABC, PK-4000, 1 Kit)
    • Follow instruction for standard reaction
  5. 3 x 10 min TBS (10x) washes
  6. DAB reaction ( reaction is slower than Parvalbumin stain, let sections stain for approximate 15 min)
    • 10% 10X TBS
    • 5% DAB (Sigma Aldrich, 3,3′-Diaminobenzidine tetrahydrochloride hydrate D5637- 1g) stock solution
    • 0.3% Ni2Cl (Sigma Aldrich, Nickel(II) chloride hexahydrate, 31462) stock solution
    • 0.05% H2O2 (30%)
  7. 3 washes in 1 x PBS after desired staining is achieved- wash in each buffer for approximately 1 min.
  8. Dehydrate in ethanol, clear in Hemo-De, cover slip with permount (Fisher Chemical Permount™ Mounting Medium, SP15-500).

NeuN staining

Day 1

  1. 3 x 10 min 1 x PBS washes
  2. Pre- incubation/ blocking step in normal goat serum for 1h at room temp on shaking rotator. (Sigma Aldrich, Goat serum, G9023, 10ml)
    • 1 x PBS + 0.3% Triton X (100%) + 3% Normal goat serum
  3. Primary anti-body incubation for 22h at room temp on shaking rotator
    • Anti-NeuN (Rabbit) diluted 1:2000 in PBS-TX (Millipore, Anti-NeuN (rabbit polyclonal), ABN78, 100ug)

Day 2

  1. 3 x 10 min PBS-TX washes
  2. Secondary anti-body incubation for 22h at room temp on shaking rotator
    • Anti- Rabbit alexa flour 405 diluted 1:500 in PBS-TX (Invitrogen, Alexa Fluor® 405 Goat Anti-Rabbit IgG (H+L)*2 mg/mL*, A-31556, 0.5ml)

Day 3

  1. 2 x 10 min PBS-TX washes
  2. 1, 10 min 1 x PBS wash
  3. Mount in 1 x PBS cover slip with vectashield mounting medium (Vector Laboratories, H-1000, 10ml).

Nissl stain

Recipe

0.5% Cresyl Violet stock solution
600ml Nanopure H2O
3g Cresyl Violet (Sigma Aldrich, Cresyl Violet acetate, C5042, 10g)
Approximately 0.6ml of glacial acetic acid to reach pH 3.5
Filter with #4 Watman paper (takes long time).

Procedure

100% ethanol – 10 min
95% ethanol – 5min
70% ethanol – 5 min
Nano pure H2O – 5 min
0.5 % cresyl violet – 6 min
Nano pure H20 – 0.5 min
Nano pure H2O – 0.5 min
Destain (2-3 drops of acetic acid in 70% Ethanol)- 15 min (or until desired colouring is achieved.
95% ethanol – 2 min
100% ethanol – 5 min
100% ethanol – 5 min
Hemo-De- 10 min
Hemo-De- until cover slipped with permount (Fisher Chemical Permount™ Mounting Medium, SP15-500).