Qualitative analysis of ß-galactosidase activity in dissected tissues

Qualitative analysis of ß-galactosidase activity in dissected tissues.

The following procedure is based on Zink and Paro, EMBO (1995) 14:5660-5671 and was adopted by Chris Bell. We have been using it primarily on salivary glands, but the procedure should be suitable for any dissected tissues. I modified this description of the protocol on 1/20/99 to use gluteraldehyde because much stronger staining was observed than when formaldehyde was used.

The following stock solutions are needed:

1% Gluteraldehyde in 1X PBS (Phosphate buffered saline).

Note that we used to use 3.7% Formaldehyde but this seems to inhibit the ß-galactosidase. In our hands, substantially better staining is obtained when the tissues are fixed with gluteraldehyde.

0.1 M NaPO₄ solution pH 7.2

Combine 28 ml of 0.2M NaH₂PO₄, 78 ml of Na₂HPO₄, and 100 ml H₂O (derived from Current Protocols in Molecular Biology)

Stain solution: combine the following and adjust to a final volume of 50 ml with water.

Amount per 50 ml	Final concentration
5 ml 100 mM NaPO₄ (pH 7.2)	10 mM
1.5 ml 5 M NaCl	150 mM
50 ul 1 M MgCl₂	1 mM
0.1267 g K₄[Fe^II(CN)₆]	6 mM
0.0988 g K₃[Fe^III(CN)₆]	6 mM
150 ul Triton X 100	0.3%

Note that the 1% Gluteraldehyde/1X PBS, 0.1 M NaPO₄ solution pH 7.2 and Stain solution (without X-gal) should be stored in the refrigerator.

Prepare a fresh batch of Stain solution with X-gal as follows:

Warm 1 ml of Stain solution to 37^oC for 5’.
Add 100 ul 20 mg/ml X-gal (originally dissolved in DMF and stored at -20^oC)
Warm at 37^oC for 5’ more and then store at room temperature while doing the assay.

Dissect salivary glands from 3^rd instar larvae in PBS. Transfer glands from several larvae to 100 ul of PBS in well slide.

It is a good idea to have a positive and negative control. Our -194 hsp70 lines can serve as a positive control. Place several larve on a moist paper towel in a small petri dish and heat shock in the air incurbator at 37^oC for 1 hr. Any nontransformed fly line such as yw can serve as a negative control.

Carefully remove PBS from glands with a pipette and immediately add 100 ul of PBS/1% Gluteraldehyde. Incubate for 15’.

Carefully remove PBS/1% Gluteraldehyde with a pipette and wash glands twice with 100 ul of PBS. Remove the wash solution with a pipette.

Put 50 ul of Stain solution w/ X-gal into a well of a 96 well microtitre dish.

Using forceps, transfer the glands to the Stain solution w/ X-gal in the microtitre dish.

Incubate at room temperature until desired level of staining is observed. For the heat shock induced -194 hsp70 constructs, color develops within 1 hour. For extended times of incubation, cover the well with tape in order to reduce evaporation.

Stop staining reaction by washing the glands with PBS.