[Histonet] RE: Immunohistochemistry TAT

Wed Jul 3 19:38:06 CDT 2013

Good as long as the wax has a chance to roll off the slide.
We tend to use a drying oven.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: Karlisch, Patricia [mailto:pkarlisch <@t> hmc.psu.edu] 
Sent: Wednesday, 3 July 2013 11:35 PM
To: Tony Henwood (SCHN); 'Sebree Linda A'; 'histonet <@t> lists.utsouthwestern.edu'
Subject: RE: Immunohistochemistry TAT

Tony,
  Great information!  We will consider keeping the dewaxing time in mind.  What do you think of hotplate warming?
Pat

-----Original Message-----
From: Tony Henwood (SCHN) [mailto:tony.henwood <@t> health.nsw.gov.au] 
Sent: Tuesday, July 02, 2013 7:08 PM
To: 'Sebree Linda A'; Karlisch, Patricia; 'histonet <@t> lists.utsouthwestern.edu'
Subject: RE: Immunohistochemistry TAT

Yes, I agree with most.

But remember a major part of dewaxing sections is to heat the sections to remove as much wax as possible. 
This allows the xylene or hydrocarbon dewaxing to be as efficient as it can.
Remember to be wary of drying temperature, too high and some antigens (eg S100, 5D3, CMV) will be adversely affected (see Henwood, A., (2005) "Effect of Slide Drying at 80°C on Immunohistochemistry" J Histotechnol 28(1):45-46).

We have noticed that staining for S100 was weaker on sections that had not been heated for as long as our routine time (30minutes 64oC).

One wonders whether heated detergent dewaxing might be more efficient at removing wax (see Henwood, A. F., Prasad, L., & Bourke, V. M. (2013). "The application of heated detergent dewaxing and rehydration to techniques for the demonstration of fungi: a comparison to routine xylene-alcohol dewaxing" J Histotechnol 36(2):45-50)

It is also possible that different waxes (especially in older, "cured" blocks) have different de-waxing requirements.

Something to consider!

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Sebree Linda A
Sent: Tuesday, 2 July 2013 10:42 PM
To: 'Karlisch, Patricia'; 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] RE: Immunohistochemistry TAT

Pat,

Right off the bat I can see 2 things that could speed things up for you.  We have Ultras and only dry our slides for 10 minutes at 60 d C.  If tissues don't stay on well, try some "Stay On" in your water bath; we're experimenting with that now.  Also, we spend way too much time ourselves searching for blocks.   See if there is a way to have them organized immediately after sectioning the H&Es so they are quicker to find...we are struggling with this.

My 2 cents; good luck.

Linda A. Sebree

University of Wisconsin Hospital & Clinics IHC/ISH Laboratory
600 Highland Ave.
Madison, WI 53792

(608)265-6596
FAX: (608)262-7174
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Karlisch, Patricia
Sent: Monday, July 01, 2013 4:36 PM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Immunohistochemistry TAT

Histonetters,
    I am looking for some help in getting IHC slides out in an 8 hour period of time.  Here is some information.

*         We have a busy Immunohistochemistry lab  and the volume of requests keep increasing.  We offer approximately 150 different  IHC stains ( including ISH and Dual ISH for Her 2).  Generally, there are request for  >150- 200  IHC tests twice a day, not including negative and positive (same slide) controls.

*         The techs pull an AM log at 6am and another at 1pm.  The 1pm log will be handled while waiting for the IHC's to be completed from the 6am run  and these will always be run overnight.

*         The 6am log requires that one tech start cutting each block for multiple IHC stains.  (A search for blocks and the necessary control slide occurs by the same person).   The slides are kept in a 60 degree oven for about 60 minutes.  Most of these slides will be ready by 2-3:30pm.
   Here is my question.  If we allowed requests for IHC stains to wait until  9AM  how could we still get slides out by 3:30pm-4pm? In other words we would not have access to an LIS log until 9am so that the pathologists could order from the morning biopsies.  What is the best way to do this  with 3 Ventana Ultra's  and one Benchmark XT.     The blocks ( usually 40-50) need to be cut; sit in an oven and then get labeled and placed on the Ventana's.   All longer stains such as ISH would be run overnight.
                                   The following  tasks makes the 3:30- 4pm deadline difficult:

*         Searching for the blocks

*         Cutting the blocks (40-50) onto control slides + negative control

*         One hour in the oven to dry  (Can we use 30minutes?)

*         Attaching labels to 150 slides and setting up instruments

*         One microtome

*         2 techs/ some of the time
     Can you share your workflow with me if you do a similar volume of slides within an 8 hours period.  How would you staff the two IHC techs to gain the most efficiency.  As you know the fuller the instrument the longer the staining process.

   Thank you,
  Pat Karlisch, Supervisor
pkarlisch <@t> hmc.psu.edu<mailto:pkarlisch <@t> hmc.psu.edu>
Tel   717-531-6072
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