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) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
