Immunohistochemical staining was performed as described previously with a few modifications (Lin et al., 2021 (link)). Drosophila adult testes were dissected in 1 × phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde for 20 min. After washing with PBST (0.3% Triton in 1× PBS) three times, the testes were incubated in a blocking buffer (5% goat serum in 0.1% PBST) for 1 h. Next, the blocking buffer was replaced by primary antibodies mixed in PAT (1% BSA, 0.1% Triton and 0.01% sodium azide in 1× PBS) and the incubation lasted for overnight. Then, the testes were washed three times with 0.1% PBST and incubated in secondary antibodies mixed in 0.1% PBST for 1 h. After washing with 0.1% PBST three times, the tissues were stained with DAPI for 10 min and washed three times before mounting with Fluoromount-G. DAPI (#MBD0015, Sigma-Aldrich, Germany), DCP1 (#9578S, Cell signaling, USA).
Dcp 1
Manufactured by Cell Signaling Technology
Sourced in Germany
Dcp-1 is a laboratory product offered by Cell Signaling Technology. It is a protein that plays a role in cellular processes. The core function of Dcp-1 is to participate in the regulation of gene expression and cellular development, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Fluoromount g, by Merck Group (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Cy3 and cy5, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Asp216, by Cell Signaling Technology (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dabco mowiol 4 88, by Merck Group (1 mentions)
4 protocols using dcp 1
1
Immunohistochemical Staining of Drosophila Testes
2
Immunohistochemistry Embryo Staining
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For immunohistochemistry embryos were fixed and stained according to standard procedures. Guinea pig anti-SIMU (30 (link)) and guinea pig anti-Drpr (32 (link)) were used at a 1:5000 and 1:100 concentrations, respectively. Rabbit anti-activated caspase 3 (Dcp-1) (Cell Signaling) and mouse anti-GFP (Roche) were used at 1:100 concentration. Rabbit anti-Crq antibody (1:500) is a gift from N. Franc. Rabbit anti-Srp antibody (1:100) is a gift from J. Casanova, K. Campbell and N. Martin. Rabbit anti-Peroxidasin antibody (1:2000) is a gift from Jiwon Shim. Fluorescent secondary antibodies (Cy3/and Cy5/Jackson ImmunoResearch; Alexa Fluor 488/Molecular Probes) were used at 1:200 dilutions. For TUNEL labeling embryos were re-fixed, washed and labeled with the In Situ Cell Death Detection kit (Roche) according to the manufacture instructions. Images were acquired on a confocal microscope Zeiss LSM 700 or on a Zeiss Axio Observer microscope equipped with an Apotome system using the AxioVision software. 75% Glycerol solution was used as the imaging medium.
Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2–3% egg volume of LysoTracker (Molecular Probes) as described in Ref. (33 (link)). Recording started 30 min following injection.
Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2–3% egg volume of LysoTracker (Molecular Probes) as described in Ref. (33 (link)). Recording started 30 min following injection.
3
Immunofluorescence Analysis of Eye Primordia
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Fixation and immunofluorescence of eye primordia was done as in [56 (link)]. Imaging was carried out on a Leica SPE confocal setup. Images were then processed using ImageJ. Primary antibodies used were rabbit anti-phospho-histone H3 (pH3) used at 1/1,000 (Sigma), rabbit against the cleaved (active) form of Dcp 1 (Dcp-1) used at 1/200 (ASP216, Cell Signaling Technology), and rabbit anti-cleaved (active) caspase 3 (Cas3*) used at 1/500 (D175, Cell Signaling Technology). Fluorescently labeled secondary antibodies were from Molecular Probes and used at 1/1,000. Rhodamine-phalloidin (Life Technologies) was used at 1/400 in some experiments to counterstain the tissue. DAPI (4′,6-diamidino-2-phenylindole, 1/10,000) was used in some experiments to counterstain nuclei. All primary and secondary antibodies were diluted in PBT (PBS + 0.1% Triton X-100).
4
Immunofluorescence Staining of Drosophila Tissues
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EADs and wing imaginal discs dissected from third instar Drosophila larvae (7 days AEL) were fixed for 25 min with 4% paraformaldehyde in PBS containing 0.1% Triton X (PBS-T) at room temperature. Adult female intestines were fixed with 4% paraformaldehyde in PBS for 48 h at 4°C. Fixed tissues were washed three times with PBS-T, intestines in PBS only. Primary antibodies were diluted in blocking buffer (PBS-T with 0.3% BSA) and tissues were stained overnight at 4°C. The following primary antibodies were used: rabbit anti-Drosophila cleaved Death caspase 1 (Dcp-1, 1:500, Cell Signaling Technology Cat #9578, RRID:AB_2721060 ), rat-anti Ecd N-term (1:500, 22 (link)), mouse-anti Armadillo (Arm, 1:20, Developmental Studies Hybridoma Bank #N2 7A1; RRID:AB_528089 ), mouse-anti disc large (Dlg1, 1:100, Developmental Studies Hybridoma Bank #4F3; RRID:AB_528203 ). After washing, the samples were incubated with the corresponding Cy3- or Cy5-conjugated secondary antibodies (1:1000, Jackson ImmunoResearch Labs Cat# 711-175-152, RRID:AB_2340607 , Cat# 712-165-150, RRID:AB_2340666 , Cat# 715-165-151, RRID:AB_2315777 ) for 2 h at room temperature and counterstained with DAPI (1:1000 dilution of 5 mg/ml stock, Carl Roth GmbH Cat# 6335.1) to visualize nuclei. Tissues were mounted on glass slides in Dabco-Mowiol 4–88 (Sigma-Aldrich Cat# D2522 and Cat# 81381) and imaged within 72 h.
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