[Histonet] crustacean haemolymph

From:"Julien Lambrey de Souza"

   Hello all histonetters,

   A  post-doc  working  in  a the next door lab is having a few problems
   with  his haemolymph smears. He is stuck with a cristalising matrix of
   haemolymph  through  wich  it  is  difficult  to  see the cells. Would
   anybody have a clue to what may be the cause? Here is his message:

   I'm  preparing  slides  from crustacean haemolymph (isopods, amphipods
   and   decapod  shrimp).   I've  been  advised  that touching a drop of
   haemolymph   to  a  slide,  followed by simple air-drying, is a method
   that should work  fine.
   However,  following  this method, the haemocytes appear to be embedded
   within  a kind of crystalline matrix.  This matrix varies in thickness
   and  in  the degree to which it obscures the cells, but it is always a
   problem,   and  remains  so  through  fixing,  staining and associated
   rinses.   If  the  haemolymph is not smeared, the crystals appear very
   thick, and cells  are suspended within the matrix.
   Any  ideas on what the problem may be with crustacean haemolymph?  I'm
   about  to  attempt  centrifugation of haemolymph + TA buffer, followed
   by   a TA wash and resuspension, but failing that, I'm a bit stuck, so
   any  ideas or suggestions of things to try are very welcome.
   The basic steps:
   Slide  prep;  drop  of haemolymph touched to a slide, then smeared (or
   24h fixation in MFA (85:10:5 methanol, formalin, acetic acid)
   10 min tapwater rinse
   hydrolysis in 5.0 N HCl
   dip in 0.1 N HCl
   120 min stain in Schiff's reagent
   3 x 5 min bisulphite rinses
   10 min tapwater rinse
   3 x 2 min dH2O rinses
   air dry, store in dark
   There  it  is.  So  if anybody has any suggestions, I will be happy to
   follow them through to him.



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