[Histonet] IHC staining problem with 30 um mouse lymph node frozen section

[msandova <@t> nd.edu]

I'm curious, why are the sections 30um thick?
Mayra




Quoting BIN MA <binma2 <@t> web.de>:

> p {margin: 0px}Dear All,   I have a problem with IHC staining(using
> Perioxdase,AEC substrate)) on 30 um  thick mouse lymph node  frozen sections.
> After staining part of section is  washed away and the morphology of the
> section is poor. Can  someone help me? My protocol is following: 
>                                IHC  staining protocol Cut 30 um frozen
> sections.Fix in cold methanol  for 3 minutes, then in cold aceton for 3
> minutes.Air dry for 1 hour.Put the section in PBS for 10 minutes.Block with
> 1%FCS/PBS  100 ul for 20 minutes at room  temperature.Endogenous biotin
> blocking:(1)   Incubate slice in egg solution for 15 minutes.Egg solution: 25
> ml egg white ,75 ml distilled water.Wash with 2 changes of  distilled
> water.(2)   Incubate slice in the milk(fat free) for 15 minutes at room
> temperature.   Wash with 2 changes of distilled water.Add diluted first
> antibody.For example :CD3-biotin Incubate 2 hours at room temperature or at 4
> °C   overnight.Wash in PBS for 30 seconds, 3  minutes and 10 minutes three
> times.endogenous peroxidase blocking:The solution : 0,3%  H2O2  ,0,25% NaN3  
> in PBS Incubate the section in this solution for half  hour. Rinse with water
> for 2 minutes. Wash in PBS  for   5 minutes.8        Block with 1%FCS/PBS for
> another 20 minutes at room temperature.9. Add   second antibody   for example
> streptavidin-PO            Incubate for 4 hours at 4 °C..     Wash with PBS
> for 1 ,3 ,10 minutes for three times.9        Add substrate 300 ul for each
> section, incubate at 37°C surface for half  hour.Substrate solution:    25 ul
> AEC stock solution                                  1 ml Na-Acetate
> buffer(5,4)                                   1ul H2O2Attention: Make this
> solution just before use,protect it from light..Wash in PBS for 30 seconds,3
> minutes   and 10 minutes for three times.10    Dry on 37°C surface for 5
> minutes.11      Mount with  175 ul Kaser’s galatine using 24x60 cover 
> glass. 12    Read the slides.  Will  adding Triton  or tween 20  help ? Thank
> you.Bin maGerman National research Centre
> ofBiotechnologyBraunschweigGermany                       
>
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