Immunohistochemistry / Hematoxylin staining

The following procedure involves transferring the slides from one solution to the next, in sequential order.

Step 1: Deparaffinizing the slides

a) Xylene (7 min)

b) Xylene (5 min)

c) 100% EtOH (2 min)

d) 100% EtOH (2 min)

e) 1% H₂O₂ in MeOH for (20 min)

f) 95% EtOH (3 min)

g) 95% EtOH (3 min)

h) 80% EtOH for (1 min)

i) 2 x (Tap water rinse for 5 min.)Prepare 1mg/ml Trypsin, 37° C

j) PBS rinse (2 min.)

k) PBS rinse (10 min.)

Step 2: Trypsinizing and antibody staining

a) Transfer slides to a tray with a lid; place water in tray to prevent drying of slides.

b) Dry surrounding areas of slide with gauze, careful not to touch sample.

c) Add 100mL of (1mg/mL., 37°C) trypsin to each slide. Incubate try for 15 min at room temperature (RT) with lid covering tray.

d) Rinse well with distilled water (5 min).

Prepare 1° antibody

e) Wash in PBS (1 min).

f) Incubate at RT in normal blocking serum (20 min).

g) Blot excess serum onto KimWipe.

h) Add 1° antibody, incubate (30 min).

i) Wash slides in buffer (5 min); dry slide NOT sample.

j) Incubate in 2° antibody solution (30 min).

Prepare Vectastatin ABC Reagent (incubate 30 min)

k) Wash slides in buffer (5 min); dry slide NOT sample.

l) Add Vectastatin ABC Reagent (30 min). (Prepare DAB solution)

m) Wash slides in buffer (5 min); dry slide NOT sample.

n) Incubate in DAB substrate solution:

o) it should take 2-10 min.

p) until the sample has a sufficiently dark (brown) color.

q) Rinse in ddH20 3X (5 min. each).

Step 3: Staining with haematoxylin

a) Rapidly dip the slides in Hematotoxylin (0.5 seconds).

b) Transfer to ddH₂O bath, and was (2 min).

c) Leave under running tap water (15 min).

d) Transfer to ddH₂O bath until next step.

Step 4: Dehydrating

a) 95% EtOH (1 min)

b) 100% EtOH (1 minute; use fresh EtOH)

c) 100% EtOH for 30 seconds

d) Xylene (1 minute; use fresh xylene)

e) Xylene for 30 seconds

f) Leave slides in Xylene until mounting.