RE: Factor X111a Protocol

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From:"Hewlett Bryan (CMH)" <HEWLETT@EXCHANGE1.CMH.ON.CA> (by way of histonet)
To:histonet <>
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Regarding your query about FactorXIIIa.

We use the polyclonal(rabbit) antibody from Calbiochem, diluted 1:1000
following trypsin digestion(0.5%w/v porcine trypsin in TBS +CaCl, 30
min@37C). Incubation in primary is 1hr@R.T, followed by standard LSAB,
chromogen is AEC.

As you know the type and amount of pretreatment will vary with the
fixation parameters.
In our initial studies we used skin fixed in NBF(0.4%w/v formaldehyde in
0.05M phosphate buffer pH7.4) for 24hrs. for shorter/longer fixation
times the pretreatment will be different and will have to be tested.
Other fixatives(Bouin, B-5 etc) may well require totally different or no
Many laboratories don't adopt rigid criteria for IHC, but merely go with
the current HOT thing (e.g HIER)!!
Validation of the results of this pretreatment often are not carried
out, as long as there is staining it is assumed all is well. This
probably explains the variation in the literature.

What I can tell you, is the result of our validation study. We perform
this type of study on all new antibodies that we use.

The following ( standardized by us) pretreatments were tested for the
Antibody protocol given above;

1) None - gave weak patchy staining.
2) Bromelain - increased background, good staining 4+.
3) Ficin - Background relatively clean, staining 3+.
4) Trypsin - Background clean, staining 4+ most complete staining.
5) Pronase E - Background patchy, staining patchy 2+.
6) Pepsin - No staining.
7) 1N HCl - background clean, staining 3+.
8) HIER in 6 different buffer systems - All gave No staining.

Hope this is of some help.



>From: 	Dave Tacha[]
>Sent: 	October 8, 1998 10:30 AM
>Subject: 	Factor X111a Protocol
>We've been using a polyclonal antibody to the A-subunit of human
>coagulation Factor XIII (Factor XIIIa).  It seems to work fine without
>any pretreatment.  We used skin, placenta and a capillary hemangioma for
>controls.  We tried digestion and it didn't seem to make much
>difference.  However, the literature shows many procedures using either
>trypsin or antigen retrieval methods.  Could someone who has work a lot
>with this antibody please enlighten me?

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