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Copyright © 2004 Cascade BioScience, Inc. All rights reserved.

IHC Protocol using Anti-PTEN, clone 6H2.1 (Cat. # ABM-2052)

I. Reagents

A. PTEN Antibody:

Anti-human PTEN clone 6H2.1, Cascade Bioscience (Cat. #ABM-2052), dilute 1:300 in PBS pH 7.4 immediately before use

B. Buffers:

PBS, pH 7.4 without Mg or Ca ( GibcoBRL cat.# 70011-044) dilute 10X PBS stock
1 part : 9 parts di water

Citrate buffer, pH 6.0. Measure 2.1g of citric acid monohydrate (Fisher A104-500) into 1 liter flask. Dissolve in 970 mL of di water. Adjust pH to 6.0 with NaOH

C. Xylene

D. 100% Ethanol

E. 95% Ethanol

F. 70% Ethanol

G. 3% hydrogen peroxide in methanol (20 mL H2O2 + 180 mL methanol)

H. Protein Block (DAKO cat.# X0909)

I. ABC Elite Kit (mouse IgG) (Vector Labs cat.# pk6102)

J. Superfrost Plus Slides, (Fisher cat.# 12-550-10)

K. Liquid DAB-Plus Substrate Kit (Zymed Labs; cat.# 00-2020)

L. Methyl Green; ready to use (DAKO cat.# S1962)

M. Permount (Fisher cat.# SP15-100)

II. Procedure

A. Cut tissue section 4-5 m thick, transfer section into a room temp water bath, and transfer section into a 48 C waterbath and adhere to slide (Superfrost Plus)

Dry slides at 37 C overnight
Bake tissue sections in oven at 60 C for 2 hours
Slides must be used within 4 days

Each case requires: 1 H&E stained section, and 1 PTEN IMPOX, 1 negative control.
Label slides appropriately (i.e. PTEN 1:300, (-) contol, H&E)

Place slides in plastic (24-slide capacity) vertical carrier. Next five steps occur in a 250mL green vertical bin

B. Deparaffinize and rehydrate tissue sections through xylene and graded ethanol series:

1. Xylene: three changes at 5 minute each
2. 100% EtOH: two changes at 5 minutes each
3. 95% EtOH: one change at 5 minutes
4. 70% EtOH: one change at 5 minutes
5. ddH20: two changes at 5 minutes each

note: H&E slides are either left in water or pulled for staining at this point

C. Quenching of endogenous peroxides (20mL of 30% H2O2 +180mL MeOH)
1. Treat sections with 3% H2O2 in MeOH for 20 minutes
2. Place in tap water bath for 5 minutes

D. Antigen retrieval (microwave): slides are to be evenly spaced in the vertical carrier; only on green plastic 250mL bin can be microwaved at a time

Microwave antigen retrieval:

1. Place slides in 200mL citrate buffer, pH 6.0 @ RT.
2. Put bin in microwave, and insert temperature reading probe into bin
3. Secure lid to the bin with a rubber band
4. Set microwave at 199F (about 93 C)
5. Microwave slides at 199F for 30 minutes, without opening door, agitating or replacing buffer.

E. Remove bin from microwave, and cool in a running water bath for 15 minutes. Then place rack ito distilled water bath for 5 minutes, and place in PBS bath for 5 minutes.

F. Remove slides from rack, dry around tissue sections with a kimwipe, and ring tissue with a pap pen smear (Zymed labs). Tissue must never dry out. Place each slide in a humidity chamber as it is prepared.

G. Blocking:

1. cover tissue sections with Protein Block (DAKO X0909)
2. Incubate slides for 10 minutes in humid chamber, room temp

H. Primary antibody incubation, by protocol as follows (either for 1 hour at room temperature; or overnight at 4 C with slides in a humid chamber)

**Note: primary antibody is appled individually to each slide after decanting and tapping edge of slide to remove excess blocking reagent.

1. place stock Ab on ice
2. 1:300 dilution of new PTEN (clone 6H2.1: Cascade BioScience)
3. place slides in humid chamber
4. cover negative controls with PBS pH 7.4
5. cover tissues with Ab dilutions or PBS
6. incubate 1hr at R.T. in humid chamber, or overnight in 4 C cold room

I. Wash of primary PTEN antibody

It is crucial to separate the two groups ( - Cont, PTEN) during the initial work to avoid cross contamination. For this reason, prepare 4 separate 250mL bins with PBS pH 7.4, two for each (-) control and PTEN slide groups.

Place all PTEN slides together in one of the 250mL PBS filled bins. Repeat for (-) controls. Then transfer to secong bin and allow to sit for 10 minutes.

J. Detection

1. Drain off excess buffer, wipe slides carefully around tissue sections, place slides in humid chamber, add diluted biotinylated IgG secondary antibody (Vector Laboratories cat.# PK6102): 1 drop of concentrated biotinylated IgG to 10 mL PBS, pH 7.4.
2. Incubate for 30 minutes at room temperature in humid chamber
3. Make up ABC reagent (2 drops of reagent A to 10mL PBS pH 7.4, mix immediately, then add 2 drops of reagent B to same mixing bottle. Mix and allow vectastain elite ABC reagent to sit 30 minutes before use.
4. Wash slides for 5 minutes in PBS buffer
5. Drain off excess buffer, wipe slides carefully around tissue sections, place slides in humid chamber. Apply vectastain ABC. Incubate for 30 min @ RT
6. Remove secondary antibody by placing all slides into two PBS bins for 10 min

K. DAB Substrate Chromogen: use Liquid DAB-PLUS Substrate Kit (Zymed Laboratories; cat.# 00-2020) in HOOD w/ gloves
***Trojanowski, J.Q. et al. J Histochem.Cytochem. 31:1217, 1983.

1. Follow kit instuctions as per Zymed, as modified below
2. in humid chamber, add DAB mix and stain for 3 min
3. drain DAB into beaker, rinse slides in 2-3 changes of distilled water
(must treat all DAB containers w/ bleach)

L. Stain enhance with 0.5% CuSO4 in 0.9% NaCl (DAB Enhancer reagent)

1. cover tissue sections with enhancer for 3 min
2. rinse in water bin, 3 changes

M. Counterstain (DAKO cat.# S1962, Methyl Greeen)

1. Blot excess water from slides, Methyl Green stain for 2 min
2. Go directly to dehydration steps
3. 95% EtOH (1 dip) > 100% EtOH (6 dips 3X) > Xylenes (3 changes at 2min each) > Mount with coverslip while specimens are still wet, using Permanent aquesous medium (Fisher; Permount cat.# SP15-100)