rev:    April 10, 2003

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PARAFFIN SECTION STAINING

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (flow cytometry, neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below. All products are for in vitro research use only-not for use in or on humans or animals-not for use in diagnostics. Price/availability/specifications subject to change without notice.

-see also saponin methodology which is a novel permeabilization reagent

Those of you who are interested in antigen retrieval (antigen unmasking) should read the latest extensive review of the technique:  Shi S-R, Cote RJ, Taylor CR. Antigen retrieval immunocytochemistry: past, present, future. J Histochem Cytochem 1997: 45(3);327-343.

Updated February 3, 1997: (Pressure Cooker, Rice Steamer, Boiling Bath)
-Listed below, please find three or more differernt sample protocols for using high temperature unmasking technqiues for staining paraffin embedded sections. As you will see, there is no one method that is best for all situations. Each antibody may have its own best method and this must be determined in your own lab.

-The following three protocols (in no particular order )have been described by others either in free literature, newsgroups, or elsewhere. Please use this as a guideline in finding the method which works best for your system

METHOD A:

       Sample High Temperature Unmasking Technqiue for Paraffin Sections:

     Buffer=1mM EDTA (ph 8.0) or 0.01M sodium Citrate Buffer (pH 6.0)

rev:12/02

1) Cut and mount sections on slides coated with "apes"

2) Deparrafinize sections and rehydrate to distilled water

3) Place sections in 0.5% hydrogen peroxide/methanol for 10 minutes (or use other appropriate endogeneous peroxidase blocking procedure). Wash sections in tap water.

4) Bring 1600ml 0.01M sodium citrate buffer (pH 6.0) to a boil in a Presto stainless steel pressure cooker, using a hot plate. Cover but do not lock lid.   or 1mM EDTA* (pH 8.0) (see ab sheet for which)

5) Position slides into metal staining racks (do not place slides close together, uneven staining may occur) and lower into pressure cooker ensuring slides are well immersed in buffer Lock the lid. The air vent/cover and overpressure plug will rise.

6) When the pressure indicator valve (the large one) has risen after about 5 minutes,  Incubate sections for 1 minute.

7) Remove pressure cooker from heat source and run under cold water with lid on. When  the small valve sinks open lid and remove slides and place immediately into distilled water. DO NOT OPEN LID UNTIL THE Vent Cover Lock Drops or Pressure Indicator shows that the pressure has been released. See Instructions with your own particular heating unit/microwave/steamer etc.

8) Wash sections in PBS* buffer (pH 7.6) for 1 X 5 minutes

9) Wash sections in distilled water for 2 X 5 minutes, then wash sections in PBS buffer   for 2 X 5 minutes.

10) Place sections in diluted normal serum for 10 minutes.

11) Cover sections with primary antibody. (Optimum dilution, incubation time and   temperature must be determined for each antibody and in each lab).

12) Wash in PBS buffer for 2 X 5 minutes

13) Incubate sections in appropriate biotinylated secondary antibody for 30 minutes

14) Wash in PBS buffer for 2 X 5 minutes

15) Incubate slides in ABC complex for 30 minutes

16) Wash in PBS buffer for 2 X 5 minutes

17) Incubate slides in DAB

18) Wash in distilled water for 2 X 5 minutes

19) Counterstain with haematoxylin (if required), dehydrate, coverslip, and mount

Material:

APES (3-aminopropyltriethoxysilane) cat#A3648 Sigma

To avoid sections becoming detached, sections should be mounted on "APES" covered slides, then dried at 37 DEG C overnight followed by drying at 56 DEG C for 60 minutes.

1mM EDTA (ph 8.0): Add 0.37g of EDTA (Sigma cat#E-5134) to 1 liter f distilled water. Adjust pH to 8.0 using 1.0M sodium hydroxide.

0.01M Sodium Citrate Buffer (ph 6.0): Add 2.94g of tri-sodium citrate to 1 liter of distilled water. Adjust pH 6 using 1.0M HCL.

SUGGESTED PROTOCOL

1. Place 1 liter of room temperature distilled water in Base(5) to Hi Fill mark.

2. Insert Drip Tray (4) in Base.

3. Place Steaming Bowl (3) on Drip Tray.

At this point, select one of two options:

4a.1. Place Rice Bowl (2) in Steaming Bowl.

     2. Fill Rice Bowl with 1.5" to 2" of  (Target Retrieval Solution or Citrate Buffer)

         or place plastic Coplin jar filled with Target Retrieval Solution in Rice Bowl and fill Rice

        Bowl with 1.5" to 2" of distilled or deionized water.

    3. Place Lid (1) on top of Steaming Bowl.

    4. Set Timer (7) for 75 minutes. Equilibration of baths/Rice

        Bowl contents to 95'C  takes approximately 45 minutes.


4b. 1. Fill Rice Bowl with 1.5" to 2" of Target Retrieval Solution or place plastic Coplin jar filled with Target Retrieval

        Solution in Rice Bowl and fill Rice Bowl with 1.5" to 2" of distilled or deionized water.

      2. Heat Rice Bowl and contents in microwave oven to near boiling (8-15 minutes).

      3. Meanwhile, place Lid on Steaming Bowl and set Timer for 75 minutes. Heat until steam is generated (10-15 minutes).

       4. Remove Lid and place Rice Bowl and heated contents in Steaming Bowl. Replace Lid. Allow temperature to                        equilibrate for approximately 5 minutes.

5. Remove Lid. Place deparaffinized and rehydrated room temperature sections in heated Target Retrieval Solution. Replace Lid

6. Incubate sections for 20 minutes.

7. Remove entire Rice Bowl and contents from Steamer and allow to cool at room temperature for 20 minutes

8. Decant Target Retrieval Solution and rinse sections two to three times with room temperature buffer (DAKO'TBS,Tris- Buffered Saline, (DAKO Code no. S3001), DAKO'PBS,Phosphate Buffered Saline (DAKO Code no. S3024), etc.).

9. Proceed with staining.

Note: Monitor level of water in Base of Steamer to avoid drying out during use.

Balaton AJ,et al. Optimization of heat-induced epitope retrieval for estrogen receptor determination by immunohistochemistry on paraffin sections. Applied immunohistochemistry 1996,4[4]:259-263

Happerfield LC, et al. Assessment of oestrogen and progesterone receptor antibodies in formalin-fixed routinely processed Paraffin-was embedded tissue. Journal of Cellular Pathology 1996,1:170-178.

- Hot plate

- 1 liter glass beaker

- 0.01M citrate buffer, pH 6.0

- PBS

To make your own citrate buffer:

1.1 Stock solutions

A. 0.1M citric acid solution: Weigh out 21.0 g citric acid, monohydrate (C6H8O7.H2O) and dissolve in 1000 ml of reagent water.

B. 0.1 M sodium citrate solution: Weigh out 29.4 g trisodium citrate dihydrate (C6H5Na3O7.2H2O), and dissolve in1000 ml of reagent water.

1.2 Working solution:

Add 9 ml of Stock solution A and 41 ml of stock solution B to 450 ml of reagent water. The pH of this solution should be about 6.0 +/- 0.1

2. Procedure

Although there are different procedures for Antigen Recovery, the following has been found to work very well for most of the antigens masked in formalin-fixed, paraffin-embedded tissue sections. Antigen Recovery is not needed for frozen sections.

2.1 Optional: To eliminate endogenous peroxidase activity, treat tissue sections with 3% H2O2 in absolute methanol for 10 minutes after deparaffinization.

2.2 Wash slides with reagent water 3 times for 2 minutes each.

2.3 Put the slides in a slide rack. Place a 1 liter glass beaker (Pyrex) containing 500 ml of the working solution of citrate buffer on a hot plate.

2.4 Heat the solution until it boils.. Put the slides in a slide rack and place it in the 1 liter glass beaker. Keep it boiling for 10 minutes

2.5 After heating, remove beaker from the hot plate and allow it to cool down for at least 10 minutes at room temperature.

2.6 Rinse slides with PBS and start the immunostaining protocol.

*Note: Tissue should be mounted on silane or poly-L-Lysine coated slides.

initial testing: assuming primary antibody approx 10ug/ml, using amplified detection as ABC methods.

3 sets standard dewax

high temp release protocols

3 sets:

2 X 5 minutes in citrate buffer

0.01M Sodium Citrate Buffer (ph 6.0): Add 2.94g of tri-sodium citrate to 1 liter of distilled water. Adjust pH 6 using 1.0M HCL

3 sets

2 X 5 minutes using 1mM EDTA (ph 8.0):

Add 0.37g of EDTA (Sigma cat#E-5134) to 1 liter of distilled  water. Adjust pH to 8.0 using 1.0M sodium hydroxide.

-to 1 set of each (3 slides total), stain as usual, ABC detection

-to another set,permeabilize for 10-20 minutes with trypsin

-to another set permeabilize with 0.1% saponin (saponin to be in all buffers after blocking step, ie with antibdy during incubation and with seocndary antibody)

see if these work. clones used and type of abs of course must be optimized

-primary and secondary antibody concentrations to be adjusted after determining which release method gives best initial staining.

RDI Division of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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