Re: Specific Hb Antigen (? h'globin, etc)

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From:"J. A. Kiernan" <>
To:"B.A.Murray" <>
Date:Mon, 15 Mar 1999 01:12:15 -0500 (EST)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Sun, 14 Mar 1999, B.A.Murray wrote:

> One of the clinicians here at the hospital would like for someone to do
> a Specific Hb
> Antigen on some blocks from years ago. He says that if they can be done
> by immuno
> or any other procedure would be fine.

  If Hb is haemoglobin, there are two easy ways to stain it
  without the trouble and expense of immunohistochemistry.
  1.  Any acid (anionic) dye at a slightly alkaline pH 
     (eg. pH 8) will fail to stai most cytoplasmic proteins
      and collagen but will still stain Hb strongly. Red
      blood cells show very nicely with acid fuchsine done
      this way. Remember to wash in slightly acidified water
      and go directly to absolute alcohol to minimize
      extraction of bound dye. A few other things (Paneth cells,
      sperm tails) will also be stained if present.
  2,  Almost any peroxidase method will detect the peroxidase
      activity of haemoglobin. A traditional technique is
      the leuco-patent blue method. Diaminobenzidine (DAB) and
      hydrogen peroxide, as used in immunohistochemistry, also
      reveals the haemoglobin in erythrocytes:  often  an
      annoyance, but for you the desired result. In animal
      material there are a few other sources of endogenous
      peroxidase (granules in neutrophil leukocytes; also, in
      frozen sections of un- or weakly fixed material the
      catalase activity of peroxisomes in many cell-types
      might be detected. It can cause confusion in some
      groups of neurons in the brain.)
  In a paraffin or frozen section of something properly fixed in
  formaldehyde you should have no difficulty making preparations
  in which most or all the stained material is heamoglobin. Cut
  the thickest sections you can see through if the object of
  this exercise is to examine vasculature.

  If Hb is not haemoglobin but ther product of, perhaps, the
  _sonic_humbug_ gene (discovered in 1998 and reported in a paper
  in Nature by 89 authors, none old enough to know what haemoglobin
  is) then the above advice will be u/s. Some HistoNet subscriber
  will probably be on your wavelength; they are a wonderfully varied
                      John Kiernan
                      London, Canada.

  P.S. Linda Margraf:  Just how many histonetters are there now?
       You put out a number in the 800s more than a year ago.
       This PS will find out if you read every message right
       down to the last line. I hope you don't!

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