Still problems with our DAB peroxidase staining + Our procedure

From:Nina Malja

Hello all histonetters
I am overwhelmed by the response I have got! I appreciate this very much!
Many of you who has answered me, asks questions regarding my reagents and
prosedures... I will try answering some of the questions here:

Nina,  are you using B-5 as a fixative for these?  Cheryl

What is B-5 fixative? We fixate our smears for 6 minutes in  36 %
formaldehyde vapour and then in 0,9 % NaCl for 10 minutes before air dried.

Hi! Nina,
i'm having similar problems w/ dab staining... however, i'm doing different 
tissues. May i know from where you are getting your dab? b/cos we thought 
it's the batch that we are having... and got down another dab box, (same lot
#) still it's not staaining.... so, pls let me know from where you are 
getting yr dab.thanks

Hi Gowry. We use 3,3'-diaminobenzidine (DAB) from Sigma. It is free base
aprx. 97%. We took over the reagent from another lab, so we don't know how
old it is, but not older than from '97. The product number is D-8001, but I
think they may have changed it later on. I got many suggestions now though
of more or less ready-to-use DAB, so if we don't figure out what is wrong we
might consider changing to that..

I recently found that bone marrow has a troublesome amount of
endogenous peroxidase that is not blocked even with hydrogen peroxide
blocking (0.3% H2O2 in H20 for 10 min at RT). Often times, the bne marrow
will stain positive even without any antibodies!!! So for bone marrows, we
now use glucose oxidase blocking always and it works like a gem. This
protocol was suggested to me by Gayle Callis on Histonet and it is great!!!
I would be glad to send you what she sent me or I can send you her e-mail
directly if you are interested. Andrea

Andrea, I would love for you to send me what Gayle Callis sent you! We still
have problems with the staining and are grateful for every hint and advice
we get! 

Today our head hematologist had a look on the smears we did last Friday and
he is still not satisfied... It seems like the cells are distroyed (possibly
due to the fixation?). We did one smear fairly ok, but the neutrophiles does
not stain properly! The eosinophiles are clearly positive, but nothing
else... HELP!! What are we doing wrong?! We did this without problems untill

Now - it is easier for me to give you our procedure than answering technical
questions. My English is rather poor and limited, so I hope you will bear
over with me...

DAB-peroxidase staining of bone marrow smears

Principle:	The peroxidase staining is negative in lymphocytes, weakly
positive in some monocytes and clearly positive in granulated cells from the
granulocyte line, + some myeloblasts. The eosinophiles will become most
strongly stained. Fixation must be done in vapour of formaldehyde. Positive
DAB peroxidase colour is yellow-brownish granulas in cytoplasma.

Reagents:	Formaldehyde solution, concentrated 36%
		NaCl 0,9%
		Tris-HCl buffer, pH 7,6, 0,05 M
		a) stock: 24,2 g trishydroxmethyleaminomethane ad 1000 mL
aqua dest. 
		b) 0,2 M HCl
		Solution: mix 50 mL of a) and 38,4 mL of b) ad 200 mL aqua
		Hydrogenperoxide 3%
		3,3'-diaminobenzidine, DAB - keep in freezer (carcinogenous)
		CuSO4 0,5% - 0,5 g CuSO4 ad 100mL aqua dest.
		10% Giemsa staining solution in Sorensens phosphate buffer,
pH 6,8

Fixation:	The smears should air fixate for 24 hours first. A fresh air
fixated smear is fixated in formaldehyde vapour for 6 minutes. The smears is
put in a container with a lid/cover and a filter paper (or cement) sprinkled
with the formaldehyde solution in the bottom. The writing field is put at
the bottom preventing the preparat from contact with the formaldehyde. After
6 minutes the smears is put carefully in 0,9% NaCl (RT) for 10 minutes. Let
the smears air dry for at least 5 minutes. If it is not possible to stain
the smears the same day, the fixated smears can be stored in a freezer
(-25C) for months.

Staining:	Add ca. 5 mg DAB to 10 mL tris-HCl and mix. Let it dissolve
in a hot water bath (37C) for about 20 minutes. It will not dissolve
completely. Just prior to the staining, add 100 µL H2O2. On a water bath
(37C), put a small container (for instance a bürker container) on the water
and let it heat up. The smears must be stained one by one. Empty the
staining solution in the small container and put the smear in it with the
preparate faced down for 90 seconds. Then move the smear over to 0,9% NaCl
for 30 seconds. Then 60 seconds in 0,5% CuSO4, ten times in 0,9% NaCl and
last ten times in aqua dest. Air dry the smear aprx. 5 minutes before
continuing. It must be completely dry first.

Contrast staining: 10% Giemsa solution for 15 minutes. Wash in Sorensens
phosphate buffer (dip aprx. 10 times).

Air dry - and we are finished.

Best regards,
Nina Malja
Bio engineer / MLT
Hematology / coagulation laboratory
Oslo, Norway

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