Rabbit anti β galactosidase

Third instar larvae were dissected in PBS and fixed with 4% paraformaldehyde, 0.1% deoxycholate (DOC) and 0.3% Triton X-100 in PBS for 27 min at room temperature. They were blocked in PBS, 1% BSA, and 0.3% Triton, incubated with the primary antibody over night at 4 °C, washed in PBS, 0.3% Triton and incubated with the corresponding fluorescent secondary antibodies for at least 2 h at room temperature in the dark. They were then washed and mounted in Vectashield mounting medium (Vector Laboratories).
The following primary antibodies were used: rat anti-Ci (DSHB 2A1) 1:50; mouse anti-Mmp1 (DSHB, a combination, 1:1:1, of 3B8D12, 3A6B4 and 5H7B11) 1:50; mouse anti-β-galactosidase (DSHB 40-1a) 1:50; rabbit anti-β-galactosidase (ICN Biomedicals) 1:2000; mouse anti-Wingless (DSHB 4D4) 1:50; rabbit anti-Dpp ([67 (link)]) 1:200; rabbit and mouse anti-PH3 (MERCK and Cell Signal Technology) 1:500, rat anti-RFP (Chromotek, 5F8) 1:2000.
Fluorescently labelled secondary antibodies (Molecular Probes Alexa-488, Alexa-555, Alexa-647, ThermoFisher Scientific) were used in a 1:200 dilution. Phalloidin TRITC (Sigma Aldrich) and Phalloidin-Alexa-647 (ThermoFisher Scientific) were used in a 1:200 dilution to label the actin cytoskeleton. TO-PRO3 (Invitrogen) and DAPI (MERCK) was used in a 1:500 dilution to label the nuclei.

Immunostaining was performed as described previously^{28 (link)}. Images were captured with a Leica (Solms, Germany) DB5500 B confocal microscope. The following primary antibodies were used: rabbit anti-Dcp1 (Cell Signalling, antibody #9578) 1:200; mouse anti-β -galactosidase (DSHB 40-1a) 1:50; rabbit anti-β-galactosidase (ICN Biomedicals) 1:2000; mouse anti-Wingless (DSHB 4D4) 1:50; mouse anti-Mmp-1 (DSHB, a combination, 1:1:1, of 3B8D12, 3A6B4 and 5H7B11) 1:50; rat anti-Ci antibody (DSHB 2A1) 1:50; rabbit anti-PH3 (Millipore) 1:100 and guinea pig anti-Myc (our own lab) 1:100. Fluorescently labelled secondary antibodies (Molecular Probes Alexa) were used in a 1:200 dilution. TO-PRO3 (Invitrogen) was used in a 1:600 dilution to label the nuclei.
DHE staining was performed as in Owusu-Ansah et al.³¹ with modifications. Larvae were dissected in 1× PBS and incubated with DHE (ThermoFisher Scientific, catalogue number D1168) 30 μM for 5 min at room temperature followed by three washes of 5 min in 1× PBS. Discs were mounted in 1× PBS and images were captured with Leica (Solms, Germany) DB5500 B confocal microscope.
All the n numbers stated in the text represent individual discs of the mentioned genotypes. All experiments are replicated at least three times.