In our hands, the quality of studies of the expression of lacZ reportergenes in frozen sections is mainly limited by poor tissue conservation. We have therefore developed a method to detect beta-galactosidase activity in glutardialdehyde fixed semithin epon sections of larval, pupal and adult brains and got best results with methylene blue/borax/toluidin blue counterstaining.
The procedure was carried out as follows: Brains were dissected in 1 x PBS, fixed on ice for 30 min in 1% glutar-dialdehyde in 1 x PBS, washed 3 x 5 min in 1 x PBS and incubated at 37 ¡C over night in a staining solution containing 25 µl of 8% X-gal in DMSO per ml staining buffer (Simon et al. , Cell 40, 805-817 (1985)). The next day, the objects were washed again for 3 x 5 min with 1 x PBS and fixed for 1 h in 6,25 % glutardialdehyde in 1 x PBS at RT. They were then washed again 3 x 5 min in 1 x PBS and dehydrated (each step 10 min at RT, in 30%,50%,70%,90% and dried 2 x in 100% ethanol). A 20 min incubation period in xylene (!) at RT followed. The brains were transfered for 1 h into a 2:1 mixture of xylene and epon 812 at RT and stored over night at 4 ¡C in a 1:1 mixture of xylene and epon 812. Then the xylene was slowly evaporated for 6-10 h at RT in the fume hood. The tissues were transfered into fresh epon and stored over night in the fridge. The objects were embedded in fresh epon the next day and polymerized for 12 h at 42 oC and 36 h at 65 ¡C. Semithin 2 um sections were cut on a Reichert-Jung Ultratome. The sections were baked over night on a hot plate at 60 oC and stained with a conventional mixture of 0,05% methylenblue/0,05% borax/0,01% toluidin- blue for not longer than 30 seconds at 60 ¡C. After washing off the excess stain with aqua dest. the preparations were embedded with DEPEX (Serva).
In sections treated with this protocol it is easy to distinguish the turquoise lacZ expressing cells from the methylene blue/borax/toluidin stained cells in the background. In our case, beta-galactosidase activity could even be detected in the fine cytoplasmatic lamellae of glial cells. It is also possible to discriminate many other cell types due to their degree of counterstaining. The results are superior to that of the widely applied pyronin G counterstaining, because the methylenblue/borax/ toluidin-blue mix reveals a wealth of structural details.