Activity staining using X-gal
1) Wash 3rd instar larvae (wandering stage) in PBS for a few minutes. Dissect in PBS.
2) Dissect anterior mouth complex by grabbing larvae at each end (Use Dumont #5 forceps). Hold larva at the posterior end and pull at the anterior end holding the mouth parts with the forceps. Usually, tissue still attached to the mouth hooks contains (most) imginal discs (the wing discs get sometimes lost), the brain/CNS complex, the salivary glands, some fatbody and anterior parts of the gut. Dissect about 5 to 6 larvae for each genotype.
3) Leave all this tissue with the mouth hooks (it can be dissected at the end of the staining procedure) and fix tissue in 1% glutaraldehyde in PBS for 15 min at RT.
4) Remove fix and wash tissue with PBS for 10 min, 2 changes of buffer.
5) Prewarm staining solution without X-gal for 10 min at 37 C.
6) Add X-gal (1/30 of a 8% stock in DMSO) to staining solution and incubate tissue in this solution for several hours to overnight, use 100-200µl per reaction (genotype).
7) After staining is completed remove staining solution and rinse discs once with PBS (optional) and resuspend them in 80% glycerol/PBS. Leave tissue for a few hours in glycerol solution before mounting.
8) Transfer tissue in a drop of glycerol (with cut off yellow tip) onto slide. Dissect
away unwanted tissue (e.g. mouth hooks) before mounting under a cover slip. For
permanent storage seal cover slip with nail polish.
Comments:
Some people prefer NaPO(4)-Buffer (pH 7.4) to PBS in dissecting and wash steps.
The staining solutions are the same as for the embryos (see embryo ß-gal protocol).
Due to the small overall size of the eye disc and the small cell size in the disc, diffusion
of the ß-gal staining is more a problem in discs than in embryos.
In lacZ-lines with strong expression the following alternative staining solution can be
used; it reduces diffusion but also reduces the sensitivity of the staining.
Alternative staining solution (based on A. Fire's protocol, moidified by U. Gaul):
1 ml of solution contains:
695 µl H(2)O
1 µl MgCl(2), 1M
200 µl NaPi (0.5m, pH 7.2)
100 µl Redox. Buffer 100mM K(3)Fe[CN]6; K(4)Fe[CN]6 4
µl 1% SDS (made fresh from 10% stock)
immediately before staining add 1/30 vol of 8% X-gal
Immunohistochemical Detection
Eye imaginal disc:
Dissections are performed as described in eye disc antibody staining protocol.
The antibody incubations and staining are also virtually identical to the protocol for eye
disc antibody stainings (see specific section).
The primary antibody, anti-ß-gal monoclonal (from Promega), is used at 1/200 to 1/500
dilutions; the secondary, HRP conjugated goat-anti-mouse (from BioRad) is used at the
same dilutions. For the incubations and washes, I usually use PBT (PBS, BSA, Triton
X-100; see embryo protocols) instead of 0.1m NaPO(4), 0.1 % saponin. However, there
is no significant difference in staining intensity or background between those two
alternatives.
signal intensification; peroxidase/DAB staining;
There are several options for intensification of the signal during and after the
peroxidase/DAB staining reaction. The eye disc antibody staining protocol describes
the osmium tetroxide postfixation and the use of Co and Ni in the DAB reaction (for
details see eye disc staining protocol). I prefer to add just NiCl(2) to the DAB reaction
(no Cobalt) as described in the ß-gal detection protocol for embryos. Since this
enhancement method does not postfix the tissue, an ethanol dehydration serie, 30%,
50%, 70%, 90% and 100% EtOH, each for 10 min, is necessary to avoid shrinking of the
tissue (this is quite important because the disc is already small enough!). A postfixation
in glutaraldehyde as an alternative is only suitable for strongly stained discs, because
part of the signal fades out during this process.
The dehydrated tissue can be stored in 100% ethanol for several days. Mounting of the
discs in DPX mounting medium; is as described in the general eye disc staining protocol.