[Histonet] RE: Sequenza units for manual immunohistochemistry (Keri Colwell)

[Liebig, Tyler K.]

Hello,

This is Tyler Liebig with the Thermo Scientific IHC group.  I'm responding to Keri's questions regarding the Sequenza manual staining.  Keri you are correct that if the waste is not emptied and builds up too much in the bottom of the sequenza unit it will definitely stop the capillary action (Reagent can't flow both ways :-).  However, this can be avoided by emptying the waste regularly between runs.  

You bring up a good question though that I didn't know the answer to: "How many slides can be stained before the waste hits the slides?" I did a quick calculation below that I hope is helpful.  

Total Waste Volume of Sequenza Rack:
In a Sequenza rack the slides are held about 1.5 inches above the bottom of the tray.  This allows about 270ml of waste before the level is close touching the slides.  

Reagent use per slide:  If you stain a single slide with a long protocol then worst case scenario is about 7 steps.  (H2O2 through Counterstain with a two step polymer)
Volume of rinse buffer: This means you would rinse 8 times 2ml each (16ml total rinse buffer per slide worst case).
For the staining reagents: The recommended volume is 0.1ml per reagent and 0.5ml for chromogen.  So for 6 reagents plus chromogen that is 1.1ml and I will double it because I know some people add way more than needed.  So 2.2ml per slide worst case.

So to sum it up worst case scenario 18.2ml of waste is created per slide which means 14 slides (270/ml/18.2ml=14.8) can be stained before the waste risks touching the slides.  The rack holds ten slides total. 

In the end I recommend users play is save and empty the waste after each batch of slides stained even though most user generate much less waste than the calculations above.

I also included a link to a great description of Sequenza use on the Histonet posted by Gayle M Callis HTL/HT/MT (ASCP).  I have referenced these details frequently myself (Thanks Gayle).
http://www.histosearch.com/histonet/Jul02A/Longansweronusetechnicswi.html

Hope that helps and if you have any other questions I'm happy to help offline (contact information is below).

Thank you,

Tyler Liebig
Americas Product Manager - Immunohistochemistry
Thermo Scientific Anatomical Pathology

 (510) 979-5000 ex 31816
(510) 299-1751 mobile

Tyler.liebig <@t> thermofisher.com