[Histonet] Re: Laczko/Levai stain protocol

[Gayle Callis]

I found several pages from Hayat's book Stains and Cytochemical Staining
1993 (for EM) with original authors cited after a Google search, key words
Laczko and Levai staining protocol.   Go to this long link and read on hints
to improve staining plus the protocol. 

 

https://books.google.com/books?id=oGj7MLioFlQC&pg=PA60&lpg=PA60&dq=Levai-Lac
zko+procedure&source=bl&ots=5i96tYhIh1&sig=iXOwXifgBUsaWGjdOGya_zjOPlA&hl=en
&sa=X&ei=IjYkVcKIN5GrogStzYLQAw&ved=0CFIQ6AEwCTgK#v=onepage&q=Levai-Laczko%2
0procedure&f=false   

 

The Google search turned up many publications using this stain for
undecalcified bone in PMMA, but you would have to access the journals for
the publications, even the original publication for the stain by Laczko and
Levai.  

 

Cartilage and Bone  (Laczko and Levai, 1975)

Azure II Methylene blue Safranin O

                Tissue was fixed with mixture of gluteraldehyde and
formaldehyde 2 -4 hr @ RT.     I assume these are tiny (minced) pieces of
tissue for EM.   Bone was decalcified for 2 weeks at 4C, postfixed with
osmium tetroxide 2 -3 hr RT, embedded in Durcupan, an EM resin.   I doubt
people embedding mineralized bone in MMA/PMMA will want to fix in
gluteraldehyde, but rather NBF and not post fix in osmium tetroxide.  

 

                The stain, as seen in many cited references was used for
thin sections of bone.  You can pick up those references via Google for more
on methods and materials for undecalcified bone in PMMA/MMA. 

 

                Azure II/Methylene Blue Working Solution

                Azure II 1%                           10 ml

                Methylene Blue 1%           10 ml

                Sodium carbonate 1%      20 ml 

 

                Protocol

                1.  Stain in working solution  for 2 -5  min at 50 - 60C

                2.  Rinse in 0.5% sodium carbonate followed by distilled
water

                3.  Counterstain with 0.5% safranin aq.  3 min at 35 - 40C
and rinse with distilled water, dehydrate.  

Results

                Chromatin, nucleoli          violet blue

                Cytoplasm                           blue 

                Erythrocytes                      dark blue

                cartilage matrix                 light blue

                bone matrix                       bright red

The search found many photos of bone with this stain to give you examples. 

 Check out these links:
https://www.unizahnklinik-wien.at/en/science/tangl_laboratory/resources.php


         and geosoft.ru/stati/de%20Sanctis%202010_J%20Clin%20Perio_.pdf 

                 by M de Sanctis - ‎2010 

                Material and Methods: Eight beagle dogs received implants
randomly installed into ..... Levai. Laczko staining. Original magnification
x 2.5. Fig. 9. Most apical .

 

Good luck

 

Gayle Callis

HTL/HT/MT(ASCP)