Hi HistoNetters

Shauna McCabe asked for some advice regarding inconsistent immunostaining
after microwave antigen retrieval using a microwave pressure cooker.  It
has been my experience that the key to successful HIER, whatever the
heating method used (microwave, steamer, pressure cooker, etc) is
consistent maintenance of temperature over time-  for example, 20-40
minutes at 98°C (time at temperature) in a microwave followed by 5-10
minutes of cooling.  Unfortunately, using a kitchen microwave pressure
cooker (Nordicware or the like) in a microwave, there is no way to maintain
a constant, verifiable temperature without fluctuations.

For this reason, Milestone developed the Glass Pressure Reactor (GPR), a
microwave pressure reactor which is available as a module for the Milestone
T/T Mega Microwave Labstation.  The GPR is designed in such a way that
temperature of the antigen retrieval solution can be controlled through the
use of a non-contact infrared temperature sensor.  Antigen retrieval can be
carried out at temperatures up to 120°C reproducibly.

Additional information on the GPR and the T/T Mega can be found on the
Hacker website.

Best regards,
Steven Slap

>Hi!
>
>I am looking for some suggestions, here is my problem:
>
>I have been doing immunostains (using ABC method) for collagen IV in mouse
>mammary tissue fixed in 10% formalin and embedded in paraffin blocks. Slides
>are cut in thin sections, are deparaffinized and rehydrated, and placed in
>3% hydrogen peroxide to eliminate endogenous peroxidase. Following this,
>they are digested in for 1 hour  0.01% pepsin.  The pepsin digestion is
>coupled with antigen retrieval. This is where the problem lies. Through a
>test battery, it was determined that the best antigen retrieval method was
>to use a microwave pressure cooker with the retrieval solution being citrate
>buffer pH 6.0 (Pharmingen recipe). The slides are left under pressure in the
>microwave for approximately 10 minutes, and then sit in the antigen
>retrieval solution approximately 8-10 minutes longer while the pressure in
>the cooker drops.
>
>With this method, the immuno- stain is usually dark and intense and the
>counterstain (hematoxylin) either will not stain or gives uneven staining.
>The usually in last sentence is the problem. No matter how rigidly the
>protocol is followed, results are never exactly the same twice. Although the
>immuno-stain is great most of the time, it is sometimes weak or non existent
>in follow up experiments. The counterstain just doesn't want to co-operate
>at all. If the pepsin digestion step is eliminated, no collagen IV is
>detected and if it is increased in length or concentration the tissue is
>'eaten' away.
>
>Any comments or suggestions in this matter would be greatly appreciated.
>
>Thanks,
>
>          Shauna  McCabe
>
>Co-op research Technician
>Glycodesign Inc.
>Toronto On.
>

************************************************
Steven E. Slap
Marketing Manager & Microwave Product Specialist
Hacker Industries & Instruments
413-733-3808
http://www.hackerinstruments.com
************************************************