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.