For live imaging, guts were dissected in 1× PBS and immediately mounted in the antifading agent Citifluor AF1 (Citifluor Ltd.). For immunofluorescence, guts were dissected in PBS, fixed for 20 min in 0.1% Tween 20-PBS (PBT) with 4% paraformaldehyde, rinsed in PBT, and then incubated with primary antibodies (1:500 anti-PH3 [Millipore], 1:500 anti-Prospero [DSHB], and 1:1,000 anti-GFP [Roche]) in PBT plus 1% bovine serum albumin. Primary antibodies were revealed with Alexa 488 or Alexa 594-coupled anti-mouse antibodies (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Guts were then scanned with an Axioplot imager (Zeiss) and recomposed using the software program MosaiX (Zeiss).
Axioplot imager
Manufactured by Zeiss
The Axioplot imager is a lab equipment product from Zeiss. It is a compact and versatile imaging system designed for a range of applications in research and industrial settings. The Axioplot imager captures high-quality images and data from various sample types using advanced optical technology.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Mosaix, by Zeiss (1 mentions)
Anti ph3, by Merck Group (1 mentions)
Alexa 488 or, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Roche (1 mentions)
Dapi reagent, by Merck Group (1 mentions)
Click it aha, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cleaved dcp 1, by Cell Signaling Technology (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
4 protocols using axioplot imager
1
Dissecting and Imaging Drosophila Intestine
2
Measuring Protein Translation in Gut
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To assess the levels of protein translation in susceptible and resistant guts, we used the Click-iT AHA (L-azidohomoalanine) for Nascent Protein Synthesis commercial kit (Invitrogen). Flies were orally infected for 16 h as described above, but by adding AHA reagent at 50 μM as final concentration to the infection mix. Guts were then dissected in 1X PBS Triton 0.3%, fixed for a minimum of 30 min in PBS 4% paraformaldehyde, and finally washed with PBS Triton 0.3%. DAPI reagent (Sigma) was used to stain DNA. The R2 region56 (link) of the gut was visualized with an Axioplot imager (Zeiss).
3
Immunofluorescence Staining of Gut Tissue
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For immunofluorescence, guts were dissected in 1X PBS, fixed for 20 minutes in PBS and 0.1% Tween 20 (PBT), and 4% paraformaldehyde; then stained with primary antibody [1/100 anti-p38c (this study); 1/500 anti-PH3 (Upstate/Millipore)] in PBT+2% BSA. Secondary staining was performed with Alexa594 anti-rabbit antibodies (Invitrogen). DNA was stained with 4′,6- diamidino-2-phenylindole DAPI (Sigma). The stained gut tissue was mounted in the antifading agent Citifluor AF1 (Citifluor Ltd.). PH3 positive cells were counted along the gut with Axioplot imager (Zeiss).
4
Immunofluorescence Assay for Midgut Analysis
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For immunofluorescence, guts from 3 to 5 day old females were dissected in 1X PBS, fixed for 20 minutes in PBS, 0.1% Tween 20 (PBT), and 4% paraformaldehyde; then stained with primary antibody [1/1000 mouse anti-GFP (Roche); 1/500 rabbit anti-PH3 (Upstate/Millipore)]; 1/200 rabbit anti-Caspase 3 activity (ThermoFischer); 1/200 rabbit anti-cleaved DCP1 (Cell Signaling Technology) in PBT + 2% BSA]. Secondary staining was performed with Alexa594 anti-rabbit and Alexa488 anti-mouse antibodies (Invitrogen). Visceral muscles were stained using 1/500 Phalloidin-Rhodamine or 1/500 Phalloidin-FITC (Life Technologies). DNA was stained with 1/15000 dilution of 4’,6- diamidino-2-phenylindole DAPI (Sigma). The stained gut tissue was mounted in the antifading agent Citifluor AF1 (Citifluor Ltd.). The mitotic index was determined by counting the number of PH3 positive cells along the midgut with Axioplot imager (Zeiss). For determination of PH3 counts, at least 10 guts were counted per condition in each experiment and data was pooled from 3 independent experiments. The mean number of mitoses per midgut and S.E.M. are shown for each genotype or treatment in the graphs.
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