Larval brains were dissected, fixed, and stained similarly to previously described (Zhu et al. 2012 (link)). Primary antibodies: guinea pig anti-Ase (1:1000), rabbit anti-Dpn (1:500) rat anti-cd8 GFP (1:400 from invitrogen 13-0081-82), rat anti-tdtom (Kerafast EST203), chicken anti-GFP (1:400 from Aves laboratory GFP-1020) chicken anti-mcherry (1:400 from Novus biologics NBP2-25158). Cleaved Caspase staining (rabbit anticleaved DCP-1) (1:200; Asp216, Cell Signaling Technology 9578S). Secondary antibodies: Alexa Fluor 488 (Invitrogen A-11006), Alexa Fluor 555 (A-21428), or 633(A-21105) (Invitrogen) were used at 1:400 and DAPI staining at 1:1000. Before imaging, brains were mounted onto slides with vectashield and coverslipped. Imaging was preformed using either Leica SP5 or Leica SP8. Brain tumor images were acquired with 1-µm stacks. MARCM clones and type II lineages were acquired with 0.5-µm stacks. Imaris 5.5 software was used for volume measurement and cell counting.
Asp216
Manufactured by Cell Signaling Technology
Sourced in United States
Asp216 is a lab equipment product from Cell Signaling Technology. It is a reagent used for protein extraction and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fluorescently labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab6662, by Abcam (1 mentions)
Fluorsave, by Merck Group (1 mentions)
Ab1218, by Abcam (1 mentions)
Vectashield with dapi h 1200, by Vector Laboratories (1 mentions)
Mouse anti diap1, by Cell Signaling Technology (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Dcp 1, by Cell Signaling Technology (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab62341, by Abcam (1 mentions)
Ab6673, by Abcam (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Ab90582, by Abcam (1 mentions)
Ab92696, by Abcam (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
6 protocols using asp216
1
Immunohistochemistry of Larval Brains
2
Immunostaining of Drosophila Eye Discs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wandering third-instar larval eye discs were dissected in 1× PBS and fixed in 4% formaldehyde+1× PBS for 15 min, permeabilized in 0.3% PBS-T (1× PBS, 0.3% Triton X-100) two times for 10 min and then incubated with antibodies overnight at 4 °C in 1× PBS+10% normal donkey serum (NDS, Jackson Immunoresearch)+0.1% Triton X-100 blocking serum. The following day, samples were washed in 0.1% PBS-T (1× PBS+0.1% Triton X-100) three times for 5 min. Samples were then incubated with appropriate fluorescently labeled secondary antibodies (Jackson Immunoresearch) for 1 h in 1× PBS+10% NDS+0.1% Triton X-100 followed by 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, samples were washed five times for 5 min then mounted in FluorSave (EMD Millipore) on glass slides. All steps were carried out at RT and with gentle rocking, unless specified otherwise. Whenever fluorescent images have been compared, they have been obtained with the same acquisition and display settings.
Primary antibodies used were: anti-β-gal: DSHB 40-1a (1:200), anti-cleaved Drosophila Dcp-1: Cell Signaling Asp216 (1:500), anti-Dac DSHB mAbdac1-1 (1:100), anti-Elav: DSHB 7E8A10 (1:200), anti-GFP (FITC): Abcam ab6662 (1/1000), anti-Hairy: from T. Orenic (1:4), anti-Hth: from Richard Mann (1:2000), anti-Notch DSHB C458.2H (1:100), anti-Repo: DSHB 8D13 (1:500), anti-Senseless: from H. Bellen (1:100).
Primary antibodies used were: anti-β-gal: DSHB 40-1a (1:200), anti-cleaved Drosophila Dcp-1: Cell Signaling Asp216 (1:500), anti-Dac DSHB mAbdac1-1 (1:100), anti-Elav: DSHB 7E8A10 (1:200), anti-GFP (FITC): Abcam ab6662 (1/1000), anti-Hairy: from T. Orenic (1:4), anti-Hth: from Richard Mann (1:2000), anti-Notch DSHB C458.2H (1:100), anti-Repo: DSHB 8D13 (1:500), anti-Senseless: from H. Bellen (1:100).
3
Drosophila Larval Tissue Fixation and Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Third-instar larvae were dissected in ice-cold phosphate-buffered saline (PBS). Collected tissues were fixed at 4% paraformaldehyde in PBS for 20 min on ice. After washing twice with PBS, fixed tissues were blocked in 5% normal goat serum/PBT (PBS + 0.3% Triton-X100) for 2–4 h at 4 °C. Samples were incubated with primary antibodies in 5% NGS/PBT at 4 °C overnight. The following antibodies were used for staining: rat anti-Ciao1 (1:100), mouse anti-GFP (1:100) (ab1218, Abcam), sheep anti-GFP (1:100) (4745-1051, BioRad), mouse anti-Elav (1:50) (from K.O. Cho), rabbit anti-BarH1 (1:100) (from J.K. Kang), rabbit anti-PH3 (1:200) (06-570, Millipore), rabbit anti-CycE (1:100) (sc-33748, Santa Cruz), rabbit anti-Cleaved Dcp-1 (Asp216) (9578, Cell Signaling), and mouse anti-Diap1 (1:100) (from B. Hay). After washing three times with PBT, secondary antibodies conjugated with FITC (1:100), Cy3 (1:600), or Cy5 (1:500) (Alexa Fluor, Molecular Probes) were incubated for at least 2 h at room temperature. After washing four times with PBT, Vectashield with DAPI (H-1200, Vector Laboratories) was used to mount the prepared samples. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope and ZEN software.
4
Immunofluorescence Analysis of Eye Primordia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
5
Embryo Immunohistochemistry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos were collected on grape juice agar plates with yeast paste. Embryos were fixed and antibody stained using standard techniques (Weavers and Skaer, 2013 (link)) with the following antibodies: anti-Cut (mouse, 1:200, 2B10-c from DSHB), anti-Odd (rabbit, 1:400, from J. Skeath), anti-Caudal (rabbit, 1:200, from Paul MacDonald), anti-Wg (mouse, 1:200, 4D4, from DSHB), anti-RFP (rabbit, 1:500, ab62341, from Abcam), anti-GFP (to visualise presence of YFP carrying balancers, goat, 1:500, ab6673, from Abcam), anti-cleaved Dcp-1 (rabbit, 1:100, Asp216, Cell Signaling technology). Secondary antibodies from Jackson ImmunoResearch of the appropriate species tagged with 488, Cy3 or Cy5 fluorophores were used. Embryos were mounted in Vectashield (Vector Laboratories) or 85% glycerol, 2.5% propyl gallate, and imaged using either a Nikon A1R or Zeiss LSM800 confocal microscope. Maximum intensity projection images were generated using Fiji.
6
Immunohistochemical Analysis of Drosophila Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Larval tissues were stained using standard immunohistochemical procedures. Briefly, discs were dissected in PBS, fixed at room temperature for 20 min in 3.7% formaldehyde/PBS and washed in 2% Triton-X100/PBS. All subsequent incubations were performed in 2% Triton X-100/PBS at 4 °C. Samples were mounted either in Vectashield or Vectashield containing DAPI (Vector Labs). The following primary antibodies were used: mouse anti-dNR215 (link) (1:100, from Ann-Shyn Chiang, mouse anti-dlg1 (1:50, 4F3, DSHB, University of Iowa, Iowa City, IA, USA), mouse anti-GFP (11814460001, Roche), mouse anti-MMP1 (1:50, 5H7B11, DSHB, University of Iowa, Iowa City, IA, USA), rabbit anti-cleaved DCP1 (Asp216) (1:200,Cell Signaling), mouse p-JNK (1:100, 9255,Cell Signaling Technology Inc., Danvers, MA, USA), PHA-555 (Phalloidin-555, A34055, Invitrogen/Molecular probes), mouse polyclonal anti-MCT1 (1:100, ab90582, Abcam), rabbit polyclonal anti-Myc (1:100, d1-717, sc-28207 Santa Cruz Biotechnology), rabbit polyclonal anti-p-PDHE1 (1:200, Pyruvate Dehydrogenase E1-alpha subunit (phospho S293), ab92696, Abcam). Note, the phosphorylation site surrounding S296 of human PDHE1 is conserved in Drosophila PDHE1, which is encoded by lethal(1)G0334 (CG7010) (e-value 5e-36, query coverage of 99%). Fluorescent secondary antibodies (1:2000, FITC-, Cy3- and Cy5-conjugated) were obtained from Jackson Immunoresearch.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!