病理小生

Immunohistochemistry

Paraffin blocks were cut at 4 μm thickness, and floated onto a water bath (45°C) before being mounted on Super Frost Ultra PlusÒ slides. The slides were then dried overnight in a 37°C oven, and overnight again in a 60°C. Slides were deparaffinised in two solutions of xylol for a minimum of 10 minutes each, then rehydrated through graded alcohols (100% and 95%) for 1 minute each before being treated in a 10% Celloidin Working Solution (see below) for one minute. The slides were then left to air dry for approximately ten minutes before being placed in 70% ethanol for 1 minute and rinsed in deionising water.

Celloidin Working Solution: 0.5% Celloidin Stock (10%) and 90% Acetone

Master Immunohistochemistry Protocol

1
Place slides in coplin jars of Tris Buffered Saline (TBS) solution (see below) for 3 minutes.

2
All the slides were placed in a Citrate Antigen (Ag) retrieval buffer (see below) in the microwave for 8 minutes after the buffer was preheated for 5 minutes.

3
Slides were cooled in Ag retrieval solution.

4
Slides were washed 3 times for 3 minutes each in TBS solution before a Peroxidase blocker (see below) was applied and developed for 20 minutes.

5
Slides were washed 3 times for 3 minutes each in TBS solution.

6
The primary antibody (Ab) was applied to each section (see section 2.3.2.1 Primary Antibodies) in 50 mL aliquots for a minimum of 30 minutes.

7
Slides were washed 3 times for 3 minutes each in TBS solution.

8
The secondary Ab was applied to each section (see section 2.3.2.2 Secondary Antibodies) in 50 mL aliquots for a minimum of 30 minutes.

9
Slides were washed 3 times for 3 minutes each in TBS solution.

10
The stain was developed in DAB+ (see below) for up to 5 minutes.

11
Slides were then rinsed in water before being counterstained in Harris’s Haematoxylin (Harris, 1900) for 4 quick dips, rinsed in water, 3 quick dips in acid alcohol solution, rinsed in water and finally treated in Scott’s Blueing solution for 30 seconds before being rinsed in water again.

12
Slides were then dehydrated through graded alcohols (70%, 95% and 100% ethanol) for 1 minute each, cleared in two changes of histolene for 2 minutes each before being mounted with DPX.

TBS solution: 0.01 M Tris, 0.15 M NaCl, 0.006% (w/v) NaN3 and 0.005% (w/v) EDTA-Na2, pH 7.4

Citrate Buffer: 10 mM Citric Acid (anhydrous), 0.05% (w/v) Tween 20, pH 6.0

Peroxidase blocker: 1/100 dilution of 30% Hydrogen Peroxide in methanol

DAB+: 1/10 dilution of DAB+ Chromagen in DAB+ Substrate Buffer (DAKO, Sydney Australia K4007)

Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0)

1. Citric acid (anhydrous)------------------1.92g

2. Distilled water-----------------------------1000ml

Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 ℃ for longer storage.