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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
-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
Sample High Temperature Unmasking Technqiue for Paraffin Sections:
Buffer=1mM EDTA (ph 8.0) or 0.01M sodium Citrate Buffer (pH 6.0)
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
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.
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
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
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
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
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
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
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
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
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
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