[Histonet] Re: Histonet Digest, Vol 102, Issue 26

[Madeleine Huey]

>> I'm a Histotechnologist working in the Regional Hospital in Barrie,
> ON Canada. We are using the Ventana Ultra for our Immunohistochemistry
> (IHC). Since the end of February, we have been having issues with some
> tissues lifting off our positive (marked with +) charged slides. It
> seems to be mostly with the fatty and/or larger sections. We now dry
> our slides for one hour at room temperature (R.T.) and an additional
> hour at 60 degrees C. We cut our IHC sections at 4 um. Since we have
> tried 2 different types of + slides and will be trying another type of
> charged slide (from Newcomer this time) I was wondering if anyone has
> any other suggestions?
>> I also have another question regarding a QC (quality control) issue.
> We use a multi-tissue control that is applied to the top of all our
> test slides for IHC. One of our paths commented that there is some
> positive staining in the smooth muscle nuclei of thenormal bowel when
> we are testing for Progesterone (PR). We are using a Heat Induce
> Epitope Retrieval (HEIR) of 36 minutes with CC1 (Ventana's proprietary
> buffer @ pH of 8.0-8.5) and a primary antibody incubation time of 16
> minutes with PR clone 1E2. (Ventana instrumentation provides
> pre-diluted antibodies and the user adjusts the concentration of the
> antibody by adjusting the time the primary antibody is incubated with
> the tissue).
>> I am concerned about the implications of this staining and I have
> not been able to find a reference to this kind of unusual staining
> pattern. The bowel tissue that we are using as QC is from a 62 year
> old female patient. I was wondering if anyone has had any experience
> with this kind of staining and /or any references that I could use.
>>
>> Thanking you in advance,
>> I look forward to your input,
>> Nancy Cloughley-Gray MLT

Nancy,

Your problem is very common, due to fatty tissue is difficult to process.

Here's my suggestion if you want to salvage your fatty tissue blocks
(presumably the fatty tissues are well fixed);

1) melt down the fatty tissue block & re-process

2) cut & put on charged slides (Fisher Scientific "Ultra Stick"
charged slides work the best)

3) bake slide for 30 min @ 60c

4) do antigen retrieval with Biocare's Diva for 3 min in Pascal or
Biocare pressure cooker (very important; do NOT use Triology & Cell
Marque pressure cooker).

5) do your ihc as usual

Three important elements; (1) well process tissue, (2) Biocare Diva AR
buffer, & (3) Pascal pressure cooker.

Keep us posted what's your outcome.

Madeleine Huey BS, HTL/QIHC (ASCP)
Supervisor - Pathology (histology & IPOX)
madeleine_h <@t> elcaminohospital.org