Larval brains (44–48 hr after egg collecting [AEC]), salivary glands (72–80 hr AEC), early (68–72 hr AEC), and late (110–120 hr AEC) wing discs were dissected in ice-cold PBS (CNS, salivary glands) or Graces medium (wing disc), fixed with 4% PFA at room temperature and probed with one or more of the following: anti-GFP (Invitrogen), DAPI (Roche), anti-HRP-Cy5 (Dianova), anti-Elav (Developmental Studies Hybridoma Bank [DSHB]), anti-Dlg (DSHB) or anti-Crbs2.8 (gift from E. Knust). The larval fat bodies (72–80 hr AEC) were fixed with 4% PFA for 30 min at room temperature and stained with anti-GFP (Invitrogen), anti-Dlg (DSHB), and DAPI (Roche). Anti-Lva (gift from E. Knust) and anti-KDEL (ENZO Life Science) were used to mark organelles. Aged (5–7 days) flies were fed for 1 day at room temperature (RT) with yeast paste; ovaries and testis were dissected and fixed with 4% PFA in PBS and stained for 3 days at room temperature with anti-GFP (Invitrogen), Phalloidin-555 (Roche), anti-Dlg (DSHB), and DAPI (Roche). All samples were mounted in VectaShield mounting medium (Vector Labs) and photographs were acquired with an Olympus1000 confocal microscope and evaluated using FIJI imaging software.
Anti kdel
Manufactured by Enzo Life Sciences
Sourced in United States
Anti-KDEL is a laboratory reagent that binds to the KDEL receptor, a protein found in the endoplasmic reticulum of eukaryotic cells. The KDEL receptor is responsible for the retrieval of proteins that contain the KDEL amino acid sequence. Anti-KDEL can be used to study the KDEL receptor and its role in protein trafficking within cells.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti mouse hrp, by Merck Group (2 mentions)
Goat polyclonal anti rabbit hrp horseradish peroxidase, by Merck Group (2 mentions)
Anti gm130, by BD (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit polyclonal anti gapdh antibody, by Abcam (2 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Anti elav, by Developmental Studies Hybridoma Bank (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti pdi, by Thermo Fisher Scientific (1 mentions)
Anti chop, by Thermo Fisher Scientific (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
Fluorochrome conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Anti glut2, by Alpha Diagnostic (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Schneider s drosophila media, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Hilymax, by Dojindo Laboratories (1 mentions)
9 protocols using anti kdel
1
Fluorescent Staining of Drosophila Tissues
2
Antibody Validation for Cellular Protein Localization
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Unless stated otherwise, reagents, buffers, culture media and serum for cell cultures were purchased from Sigma-Aldrich (Milan, Italy). Custom-made rabbit polyclonal anti-NS antibody and rabbit polyclonal anti-GAPDH antibody were from Abcam (Cambridge, UK). The mouse monoclonal anti-NS antibodies were made in-house as reported before (Miranda et al., 2008 (link)). Anti-KDEL was from Enzo Life Sciences (through 3VChimica S.r.l., Italy), anti-GM130 from BD Transduction Laboratories and anti-catalase from Merk Millipore (both through SIAL S.r.l., Italy). Goat polyclonal anti-rabbit-HRP (horseradish peroxidase) and rabbit anti-mouse-HRP are from Sigma-Aldrich (Milan, Italy). Goat anti-mouse IgG-Alexa Fluor® 488 and -Alexa Fluor® 594, and goat anti-rabbit IgG-Alexa Fluor® 594 were from Life Technologies (Milan, Italy).
3
Western Blot Analysis of ER Stress Markers
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Hippocampi samples (n = 5 per group) were homogenized in a lysis buffer containing protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 10 mM PMSF). After protein quantification by the Bradford reagent method, aliquots (30 μg) were diluted in a sample buffer, subjected to 12% SDS–PAGE, and transferred to nitrocellulose membranes. Detection of specific proteins was performed by incubating membranes with primary antibodies (anti-Kdel (1:1000, Enzo Life Sciences, Farmingdale, NY, USA), anti-Pdi (1:1000, Thermo Fisher Scientific, Waltham, MA, USA), anti-Chop (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) and anti-Parp-1 (1:2500, Calbiochem, San Diego, CA, USA), followed by peroxidase-conjugated secondary antibodies and revealed by the chemiluminescence method (peroxidase-H2O2-luminol). The results were expressed as relative density. An equal amount of protein in each lane was confirmed by hybridization with anti-β-Actin (1:1000, Sigma Aldrich, Saint Louis, MO, USA).
4
Multicolor Immunofluorescence Analysis of Mouse Pancreas
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Mouse whole pancreas tissue was fixed in 4% formalin and processed for paraffin embedding. Antigen retrieval was performed by microwave heating in antigen retrieval solution-citrate buffered (Prosan, Merelbeke, Belgium). Detection was done using fluorochrome-conjugated secondary antibodies (Jackson Laboratory, Westgrove, Pennsylvania, USA) according to manufacturer’s instructions.
The following primary antibodies were used: anti-insulin (Guinea pig Polyclonal—C. Van Schravendijk, Diabetes Research Center, Brussels, Belgium); anti-PP (Rabbit Polyclonal—R.E. Chance, Lilly Research, Indianapolis, USA); anti-Glut2 (Rabbit Polyclonal—Alpha Diagnostic—San Antonio, Texas, USA), anti-KDEL (Mouse monoclonal – Enzo Life Sciences – Farmingdale, NY).
Pictures were acquired with ZEISS LSM7 10 NLO confocal microscope using ZEN 2009 software (Carl Zeiss, Oberkochen, Germany). Quantification of insulin and PP-positive areas and pancreas tissue area (85,29±9,58 mm2 spread across 3 non-consecutive sections) was performed with IPlab 4.0 software (Becton Dickinson, San Jose, CA, USA).
The following primary antibodies were used: anti-insulin (Guinea pig Polyclonal—C. Van Schravendijk, Diabetes Research Center, Brussels, Belgium); anti-PP (Rabbit Polyclonal—R.E. Chance, Lilly Research, Indianapolis, USA); anti-Glut2 (Rabbit Polyclonal—Alpha Diagnostic—San Antonio, Texas, USA), anti-KDEL (Mouse monoclonal – Enzo Life Sciences – Farmingdale, NY).
Pictures were acquired with ZEISS LSM7 10 NLO confocal microscope using ZEN 2009 software (Carl Zeiss, Oberkochen, Germany). Quantification of insulin and PP-positive areas and pancreas tissue area (85,29±9,58 mm2 spread across 3 non-consecutive sections) was performed with IPlab 4.0 software (Becton Dickinson, San Jose, CA, USA).
5
Visualizing WFS1 Localization in Drosophila S2 Cells
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Drosophila S2 cells were maintained in Schneider's Drosophila media (Thermo Fisher Scientific) supplemented with 10% FBS in a 25°C incubator. S2 cells were transfected with actin-GAL4 and UAS-wfs1-HA plasmids using HilyMax (Dojindo) following the manufacturer’s protocol. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 48 h after transfection and stained with anti-HA (Santa Cruz Biotechnology) or anti-KDEL (Enzo Life Sciences) antibody. Antibodies were detected by anti-rabbit IgG conjugated Alexa 488 (Abcam) or ant-mouse IgG conjugated Alexa 647 (Abcam). ER and mitochondria were labeled by Concanavalin A (ConA) conjugated Alexa 647 (Thermo Fisher Scientific) and Mitotracker (Thermo Fisher Scientific) respectively. The stained cells were analyzed using a confocal microscope (Carl Zeiss, LSM 780).
6
Immunoblotting and Immunofluorescence Assays
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Unless stated otherwise, reagents, buffers, culture media and serum for cell cultures were purchased from Sigma‐Aldrich (St Louis, MO, USA). Custom‐made rabbit polyclonal anti‐NS antibody 33 and rabbit polyclonal anti‐GAPDH antibody were from Abcam (Cambridge, UK). The mouse monoclonal anti‐NS antibodies were made in‐house as reported before 9 . Anti‐KDEL was from Enzo Life Sciences (Farmingdale, NY, USA) and anti‐GM130 from BD Biosciences, San Jose, CA, USA. Goat polyclonal anti‐rabbit‐HRP (horseradish peroxidase) and rabbit anti‐mouse‐HRP are from Sigma‐Aldrich. Goat anti‐mouse IgG‐Alexa Fluor 488 and ‐Alexa Fluor 594, and goat anti‐rabbit IgG‐Alexa Fluor 594 were from ThermoFisher Scientific (Waltham, MA, USA).
7
Western Blot Analysis of Liver Proteins
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Liver samples (n = 7) were homogenized by sonication with lysis buffer containing protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 10 mM PMSF). For each sample, 30 μg of total protein was diluted with sample buffer and loaded into a SDS-PAGE gel for protein separation, which was transferred to nitrocellulose membranes. For the detection of the proteins of interest, membranes were incubated with primary antibodies: anti-KDEL (Enzo Life Sciences, USA, Cat# ADI-SPA-827), anti-XBP1 (Enzo Life Sciences, USA, Cat# ADI-905-739), anti-PDI (Enzo Life Sciences, USA, Cat# ADI-SPA-891), and anti-MTP (Sigma-Aldrich, USA, Cat# AV43618), followed by incubation with peroxidase-conjugated secondary antibodies for chemiluminescent detection (peroxidase-H2O2-luminol). β-Actin (Sigma-Aldrich, USA, Cat# A5441) was used as protein loading control.
8
Mitochondria-Endoplasmic Reticulum Interactions
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Oligomycin (O4876), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (C2920), antimycin A (A8674), rotenone (R8875), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (C2759), bafilomycin A1 (B1793), sodium pyruvate (P5280), and glucose (G7021) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). MitoTracker Deep Red (M22426) and l -glutamine (25030-024) were purchased from ThermoFisher Scientific (Illkirch, France). Cyto-ID (ENZ-51031) was purchased from Enzo Life Sciences (Villeurbanne, France). Anti-Mitofusin 2 (rabbit, 9482), anti-Grp75 (rabbit, 3593), anti-LC3B (rabbit, 2775), and anti-Myc (mouse, 2276) were from Cell Signaling Technology (Saint Quentin Yvelines, France). Anti-Tom20 (rabbit, sc-11415), Anti-Tom20-AF488 (rabbit, sc-17764), anti-Parkin (mouse, sc-32282), and anti-Ero1-Lα (mouse, sc-365526) were from Santa Cruz Biotechnology (Heidelberg, Germany). Anti-VDAC1 (mouse, ab14734), anti-Parkin (rabbit, ab15954), and anti-Beclin-1 (mouse, ab114071) were from Abcam (Paris, France). Anti-KDEL (mouse, ADI-SPA-827-D) was from Enzo Life Sciences. Anti-PACS-2 (rabbit, 19508-1-AP) was from Proteintech (Manchester, UK), and Anti-PACS-2 (rabbit, clone 18143) was previously reported [9 (link)]. Anti-IP3R1 (rabbit, 07-1213) was from Merck Millipore (Molsheim, France). Anti-β-actin (mouse, A2228) and anti-P62 (rabbit, P0067) were from Sigma-Aldrich.
9
Western Blot Analysis of iPSC-HLOs
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