Re: Luxol Fast Blue-Holmes stain

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From:"J. A. Kiernan" <>
To:Curtis King <>
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On Tue, 22 Aug 2000, Curtis King wrote:

> If anyone has a procedure for a combination Luxol Fast Blue-Holmes silver 
> stain could you please send it to me.

  Do the complete Holmes silver, with gold toning-oxalic-acid-thiosulphate
    follow-up) first. (It involves two alkaline solutions, the dilute
    buffered AgNO3 and the sulphite-hydroquinone developer. These could
    extract luxol fast blue if that staine were done first.)
      Holmes' method is very reliable once you've got it working. You
    may need to vary the pH of the borate buffer and/or halve or
    double the concentration of silver nitrate. Often you can get away
    with using his original (1943) technique in which the only silvering
    is in the dilute buffered solution, overnight at 37C. This saves
    effort, black fingers and a lot of money. Otherwise, use Holmes'
    final (1947) procedure, with the pre-treatment in 20% AgNO3 and
    a dash of pyridine in the overnight dilute solution. (This is the
    method given in most staining manuals, but the 20% AgNO3 step is
    rarely necessary. The price of silver nitrate has been ridiculously
    high for many years.)
      Silver methods for axons work well only if you understand the
    reasons for all the steps in the method, and Holmes (1943) provided
    the simplest of all these methods for paraffin sections. His paper
    in Anatomical Record 86: 157-187 is one of the all-time great classics
    of histotechnology. Better than Bodian (1936, 1937) and Samuel (1953),
    IMHO. Peters (1955) published a series of 4 papers in the Quarterly
    Journal of Microscopical Science on silver staining of axons in
    paraffin sections that firmly established the validity of the chemical
    principles (dating from Liesegang 1911 and the scientific study of
    photographic development). Little (perhaps nothing) has been added to
    knowledge of this field since 1955, but many methods, new in one way
    or another, have been described. I could say much more, but it
    wouldn't be answering Curtis King's question!
      Technical tip. Do not rinse the slides in water after the overnight
    stay in very dilute, buffered silver nitrate, before their immersion
    in the sulphite-hydroquinone (Bodian's) developer. If you understand
    how silver methods work, you will know the reason for this. If you
    don't know why you shouldn't wash away all the AgNO3 you need to
    read a textbook (or Holmes 1943, cited above) before going ahead.
      Silver methods for axons have a reputation for fickleness second
    only to that of silver methods for neuroglial cells. In a lab that
    I visit occasionally they have a framed photo of Santiago Felipe
    Ramon y Cajal on the wall, scowling down at the vessels and bottles
    on the bench. Wizardry is not dead. That lab now puts out publications
    with the best illustrations of immunohistochemical preps that you
    could ever expect to see.  

  Next do the complete luxol fast blue procedure. The reason for doing
    it after and not before the Holmes stain is given above.

  Finally, if you want the lot, counterstain with neutral red to show
    neuronal Nissl substance (cytoplasmic RNA) and nuclei. Dehydrate
    quite abruptly (3 X 100% alcohol), clear in xylene (X2) and apply
    the coverslips with any resinous mountant.

  Ideally you'll end up with black or dark red axons, many with blue
  myelin sheaths, and red neuronal cell-bodies. My experience with
  combining these methods is that pleasing results are obtained only
  in parts of the sections, and I don't think it's worth trying too
  hard. Axon (silver) plus myelin combination stains need oil 
  immersion to be properly appreciated, and even then they do little
  more than show myelin sheaths around the larger axons.

  Conclusion.  Holmes is good. So is luxol fast blue. Doing both on
    the same section may not be worth the effort

  Addendum.  Luxol fast blue isn't the best myelin stain, but it's
    probably better than others if it has to follow a silver method
    for axons.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1


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