Long answer on use technics with Sequenza Staining Racks

From:Gayle Callis

Work like a charm, can use plus charge or regular slides.

The capillary gap/space between coverplate and slide is designed to hold 80
ul volume, 100 ul is a good amount to add, although some people are using
dropper bottles, no problems. 

I numbered each slot in front and on sides 1 through 10 to know which slot
I was working at, mentally keeping track of sequence.  

Sequence of events/some How To's!

Write on slide to read top of label, nearest end - easier to keep track.
We sometimes had three different antibodies in one holder, with different
neg isotype controls, etc.  

Mount your section closer to end of slide towards plus charge marks, not
near label end.  It is better to avoid multiple sections per slide or
offset them so flow of reagents is smooth (ie like capillary gap Microprobe
system).  Frozen sections need to be handled with TLC with coverplates.  

A reagents must be ROOM TEMPERATURE, aqueous liquids de-gas as the warm up,
air bubbles happen.  

Holders can be microwaved, incubated at 37C, but if you need overnight cold
incubations, let the holders warm up to RT for approx 1 hour before adding
warm reagents to prevent bubbles . 

If you use peroxidase blockers containing high concentration of
methanol/H2O2, it may be advisable to do this step separately before
mounting coverplate, or you get bubbles. With frozen sections, the DAKO
peroxidase block did not give bubbles, blocking was done on
slide/coverplate, 2 - 3 drops per section to equal volume needed.  

Mount slide on a coverplate - wetted well with buffer or buffer containing
detergents, the slide is mounted like a coverslip as bubbles will be the
ENEMY, takes practice. 

FILL well with buffer from rinse bottle, and let it run through - they say
5 minutes.  The little plus charge marks at bottom of slide have a
thickness of approx 147 um = gap or clearance between slide/coverplate.
(Yes, I did the calculation!) Timing was closer to 4 minutes - you only
need to do this ONCE. If you do repeated rinses by filling the wells, say 3
times, the holder fills up, and reagent will touch bottom of slides, get
sucked back under coverplates, a mess. The total rinse is approx 5 mls =
approx 5 minutes, not bad 1000 ul/minute through a gap holding 80 ul! 

ANY coverplate that runs TOO fast means it is defective, is pulled, slide
remounted with new one, old coverplate tossed. 

Pipette your antibodies/blockers (consider repeating pipettors!) or use
dropper bottles (you can measure drop volume with a pipettor, 1 drop =
approx 20 ul?) 100 ul per well, we like to introduce reagent at back of
well to prevent bubbles formation, set timer and walk away.  Repeat your
rinsing, etc as you go along. WE also cut the tips on buffer squeeze
bottles to have a slightly wider hole, gives a gentler stream, and often
start the stream away from well to eliminate buffer, a little practice for

You can leave the coverplate on for chromogen, but we take off to
develop/control chromogen with microscope - at that point going to a flat
staining rack with white underneath, towels. etc. Coverplates were removed
with a bit of buffer in wells to permit a sheeting action during removal.  

Coverplates discolor, can be washed but scratches are also an enemy. Any
residual bleach used, or detergents could mess up your IHC. Extensive
rinsing is mandatory if you bleach, wash with soaps, etc. 
We wash by immersing coverplate into distilled water immediately after
removal, then rinse with tap water and another distilled water, air dry,
reuse - taking care to not scratch the surface, if so, toss it out.
Never touch surface of coverplate with your fingers, skin is oily, ruins
the flow properties! 

We can set up as many holders as we want 10 holders = 100 slides during a
morning run.  Have also used separate timers per holder or every two
holders to not overincubate. Adjustment for lag time between slides was not
a huge factor, it went very quickly. 

A good thing to do is demonstrate HOW the coverplate works.  Mount a slide
with buffer, then pipette 100 ul of hematoxylin in the well, and watch it!!
Amazing flow, also shows how/spread/direction of flow and gets rid of
mystery on what is actually going on.  A lovely efficient system, buffer
usage became minimal, less expensive. Our staining was clean, results
excellent, some report better - antibody is in close contact with section.  

Whew, what a lecture on technic late in the week, have a great weekend and
have fun with coverplates. 


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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