Primary cortical neurons (DIV 14) were treated with vehicle (DMSO) or MG132 (20 μM) for 9 h. Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS, the cells were blocked with 10% goat serum in 0.2% Triton X-100/PBS for 2 h at 37°C and incubated with rabbit anti- GABAAα1 (1:200) and mouse anti-PDI (1:1,000; Enzo Life Sciences, Farmingdale, NY, USA) overnight at 4°C. After washing and incubation with Cy3 or Cy2-based secondary antibodies (1:200), the cells were washed extensively with PBS, and mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen). Confocal images were taken with a Zeiss LSM-510 confocal microscope. Colocalization of proteins was confirmed by z stack analysis. Data show representative single plane images.
Mouse anti pdi
Manufactured by Enzo Life Sciences
Sourced in United States
Mouse anti-PDI is an antibody that specifically binds to and detects protein disulfide isomerase (PDI), an enzyme involved in the formation and rearrangement of disulfide bonds in proteins. This antibody can be used for the identification and analysis of PDI in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti actin, by MP Biomedicals (1 mentions)
Rabbit anti gm130, by Abcam (1 mentions)
Rabbit anti tom20, by Santa Cruz Biotechnology (1 mentions)
Clone tub 2, by Merck Group (1 mentions)
Mouse anti drp1, by BD (1 mentions)
Mouse anti giantin, by Enzo Life Sciences (1 mentions)
Mouse anti kdel, by Abcam (1 mentions)
Mouse anti cytochrome c, by BD (1 mentions)
Alexa 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Provis ax70, by Olympus (1 mentions)
Donkey anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
3 protocols using mouse anti pdi
1
Immunofluorescence Analysis of GABA Receptors
2
Immunoblotting and Immunocytochemistry Protocols
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mouse anti-cytochrome c (2 h, RT, 1:1000, clone 7HB8.2C12; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-TOM20 (1:200; Santa Cruz, Dallas, TX, USA), mouse β-tubulin (2 h, RT, 1:1000 in immunocytochemistry (IHC) and in western blots, clone TUB2.1, Sigma Aldrich), mouse anti-DRP1 (2 h, RT, 1:1000 in IHC and western blot; BD Bioscience, Franklin Lakes, NJ, USA) for Figure 3a/c and Supplementary Figures S3A–C , mouse anti-DRP1 (2 h, RT, 1:800; BD Bioscience) for Figure 3d , rabbit anti-p-DRP1 (1:500; Cell Signalling, Danvers, MA, USA), mouse anti-actin (1:1000; MP Biomedicals, Santa Ana, CA, USA), mouse anti-KDEL (o/n, 4 °C, 1:500; Abcam, Cambridge, UK), mouse anti-PDI (o/n, 4 °C, 1:1000; Enzo Life Sciences, Lörrach, Germany), rabbit anti-GM130 (o/n, 4 °C, 1:250; Abcam), mouse anti-giantin (o/n, 4 °C, 1:1000; Enzo Life Sciences). Generation of rabbit anti-COXII (1:1000) has been described before.22 (link) Alexa Fluor 488-conjugated phalloidin (1:500; Invitrogen) was used to visualize F-actin.
3
Immunostaining Protocol for CDNF Localization
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The parental cells or stable cell clones were seeded on coverslips, transfected in case of parental cell line, fixed with 4% PFA (48 h after transfection), permeabilized with 0.1% Triton-X and stained with following antibodies: rabbit anti-CDNF (0.4 μg/ml1), mouse anti-PDI, 1:150 (Enzo Life Sciences, Farmingdale, NY, USA), or mouse anti-GM130, 1:100 (BD Transduction LaboratoriesTM, Franklin Lakes, NJ, USA). Secondary antibodies goat anti-rabbit Alexa 568, 1:400), and donkey anti-mouse Alexa 488, 1:400 (both from Life Technologies, Thermo Scientific) were used for detection of the corresponding primary antibodies. Nuclei were stained with Hoechst 3345 (Sigma-Aldrich). Fluorescent microscopy images were captured using confocal microscope (Leica TCS SP5, Wetzlar, Germany), or epifluorescent microscope (Olympus AX 70 Provis, Tokyo, Japan).
For immunocytochemistry, following controls were included: (1) omission of the primary antibodies, for analyzing possible unspecificity of the secondary antibodies used, and (2) separate single antibody stainings, for analyzing possible channel leakage. Non-transfected wtARPE-19 cells stained with the in-house-produced CDNF antibody did not give background with the light exposure time used (seeFig. 3b , Results), reflecting low endogenous CDNF levels and high specificity of the antibody used.
For immunocytochemistry, following controls were included: (1) omission of the primary antibodies, for analyzing possible unspecificity of the secondary antibodies used, and (2) separate single antibody stainings, for analyzing possible channel leakage. Non-transfected wtARPE-19 cells stained with the in-house-produced CDNF antibody did not give background with the light exposure time used (see
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