Confocal microscopy was performed as described previously with minor modification6 (link). Briefly, cells were grown on glass cover slips to 70% confluence and were infected with 10 MOI Ad GFP or TCTP-GFP virus. Cells were incubated for 24 hours and then fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. For staining E-cadherin, cells were fixed with 100% methanol for 15 minutes at −20°C. After washing with PBS, the cells were permeabilized in 0.2% Triton X-100 in PBS and blocked in 1% bovine serum albumin. Cells were than stained with mouse monoclonal anti-E-cadherin antibody (1:200), anti-N-cadherin antibody (1:200) and rabbit polyclonal anti-β-catenin (1:200) and then probed with Alexa Fluor 488 or 594 conjugated goat anti-mouse IgG (Invitrogen, CA, USA). For F-actin staining, Cells were stained with rhodamine conjugated phalloidin (Molecular Probes, OR, USA). Counterstain with DAPI was performed by mounting in ProLong Gold antifade reagent (Invitrogen, CA, USA). Cells were photographed at × 400 magnifications with a fluorescence confocal microscope (LSM510, Zeiss, Germany). Cell morphology was examined under an inverted microscope (Zeiss, Germany) at × 400 magnification.
Alexa fluor 488 or 594 conjugated goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 or 594 conjugated goat anti-mouse IgG is a secondary antibody used in immunological assays and microscopy applications. It binds to mouse immunoglobulin G (IgG) and is conjugated to the Alexa Fluor 488 or 594 fluorescent dye for detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Inverted microscope, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ab183495, by Abcam (1 mentions)
Mab360, by Merck Group (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Af 556 na, by R&D Systems (1 mentions)
Ma1 045, by Thermo Fisher Scientific (1 mentions)
3 protocols using alexa fluor 488 or 594 conjugated goat anti mouse igg
1
Confocal Microscopy Analysis of Cell-Cell Junctions
2
Immunostaining of Pronephric Cell Structures
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Immunostaining was performed in whole-mount larvae as described previously58 (link). A mouse anti-NA-K-ATPase alpha-subunit (a5) antibody (1:200, Developmental Studies Hybridoma Bank) was used to label the location of NA-K-ATPase in the pronephric epithelial cells. A mouse anti-acetylated alpha-tubulin antibody (1:200, Sigma) was used to label the primary cilia of pronephric ducts. A mouse anti-c-myc (9E10) antibody (1:500, Covance) was used to label the Myc-tag of LipL32 protein expression. A rabbit anti-GFP antibody (1:500, Invitrogen) was used to label the pronephric GFP expression in the wt1b:GFP larvae. The secondary antibodies used were Alexa Fluor 488- or 594- conjugated goat anti-mouse IgG and goat anti-rabbit IgG (1:500, Invitrogen). The immunostained larvae were then embedded in OTC medium and cryosectioned. Sections were mounted in Vectashield (Vector Laboratories) with DAPI and observed under fluorescence microscopy.
3
Immunofluorescence Analysis of Brain Proteins
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In brief, brain sections were fixed (ice cold 4% paraformaldehyde), washed, permeabilized (0.3% Triton X‐100 in PBS) and blocked with appropriate non‐immune sera. After that, specimens were incubated (overnight, 4◦C) with primary antibodies against mouse anti‐PSD95 (1:500, mouse, MA1‐045, Thermo Fisher), anti‐GFAP (1:500, MAB360; Millipore), rabbit anti‐GFAP (1:5000, Z0334; Dako), anti‐GluN2A (1:500, NBP2‐19551, Novus Biologicals), anti‐furin (1:500, ab183495; abcam), anti‐SNAP23 (1:500, 10825‐1‐AP; proteintech), anti‐VAMP3 (1:500, 10702‐1‐AP; proteintech), anti‐pNF‐κB (1:500, 3033; CST), anti‐synaptophysin (1:500, 36406; CST), the goat anti‐β‐NGF (1:200, AF‐556‐NA; R&D Systems). The secondary antibodies used in the present experiments were Alexa Fluor®‐488 or −594 conjugated goat anti‐mouse IgG, and Alexa Fluor®‐594 or‐488 conjugated goat anti‐rabbit IgG or donkey anti‐goat Alexa Fluor®‐594 conjugated donkey anti‐goat IgG (1:500, Invitrogen). Cell nuclei were visualized with Hoechst 33342 (1:1000) or DAPI. Fluorescence‐labeled optical sections were captured with a Leica confocal laser scanning microscope (TCS SP5). Image J software (W.S. Rasband, U. S. National Institutes of Health, Bethesda, Maryland, USA) was used for immunofluorescent intensity quantitative analysis of brain sections.
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