[Histonet] Kappa and Lambda
I have no experience with Bouin, i will give it a shot and reply anyway.
As far as i know the rabbit polyclonals K/L from DAKO are good. If you are detecting K/L in plasmacells i think many monoclonals performs equal or might be better. In cells wich express low amount of K/L (mature B-cells etc)i think your polyclonals are one of the best there is.
I have some problems with K/L on bonemarrow too.
Optimal staining of K/L requires optimal fixation.
I think our problems on bonemarrow is due to how the sample is taken.
Small biopsies often gets excessive background due to squeezing of the tissue. * Further dilution 2-3x might help.
Delayed fixation might give excessive background. * Not much one can do with this??
Poor fixed samples might give heavy background staining. * Might help to reduce Antigen Retrieval with 20-35%.
I have seen optimal staining in different publications can be achieved with; proteolysis, TRS antigen retrieval, Citratebuffer pH6,0, Tris-EDTA buffer pH 7,8 and combi Citratbuffer + proteolysis.
I have tried most proteases, combi methods and buffers (including TRS-buffer). I found long citrate-buffer retrieval to be the best on our samples, HIER with citratebuffer pH 6,0 at 100degC about 30mins.
Polyclonal K (Dako) usually somewhere around 1:4000 (dependent on incubation time/temp, and sensitivity of detection system), L usually 1,5-2x higher dilution.
You might try double immunofluorescence eg L FITC / K Txr, "backgound" gets an orangecoloured staining.
I use this when detecting plasmacells on eg bonemarrow with much background (with IHC-staining)
im not experienced with different fluorecence-techniques, there probably better fluorochromes than Fitc and TxR.
Histonet mailing list
<< Previous Message | Next Message >>