You pose an interesting question, and there seems to be a great deal of
variation with times and steps, particularly when you read written
protocols on the web or what people supply on Histonet. I presume you are
talking about manual staining and not an automated stainer?
For 5 um or less frozen or paraffin embedded sections, enzyme
immunohistochemistry, I am not an excessive rinser, but have adequate
rinses with excellent results. I found it did NOT make any difference in
results if rinses were untimed or with more than one rinse. Timing rinses
was abandoned a long time ago as, sorry for the pun as too "time consuming"
. I reached a point of refusal with 3 to 5 minutes per rinse and with three
changes - it simply did not jive with an busy IHC lifestyle for a
day. Using a coplin jar for rinsing was a horror since we 20 or more
slides at a time.
For all immunostaining, we use manual Scytek humidity chambers that tip the
slides into a slanted upright position, but slides stay in place. We do
one untimed rinse between steps by flowing buffer from a wide tipped squirt
bottle so buffer flows from above and across sections, slowly and gently -
over and back one time. After rinse, buffer is either added to section
until next step (have to open tubes, or some extra manual step) or I simply
blot and add the next reagent. I based this rinse on Shandon coverplate
method, where the well is filled, buffer flows down and over sections using
a capillary gap, gravity flow. We found this takes approx. 2 -3 minutes to
completely drain the well (one should time this), and NOT the 5 minutes
they say to use.
For immunofluorescence staining, I do the same thing, but tip buffer off
for extra rinses after the fluorophore step. I tend to rinse with more
steps for IFA staining, just to get rid of the fluorophore and any glowing
Since I do not have an automated immunostainer, what kind of rinsing step
do these have? If these perform a single rinse without any particular
time, then why not emulate the machine? I figure if a machine can do it,
so can I.
At 11:02 AM 5/23/2007, you wrote:
>What are the standard rinse times in buffer that people are using between
>steps of a standard immunohistochemical procedure? For example, after the
>primary antibody, before applying the secondary? How many rinses, and of
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
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