Re: [Histonet] Rinse times during immunohistochemical staining?

From:Gayle Callis


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 
>what duration?

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610

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