Cells (3 × 104 cells per well) grown on cover slips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 3% BSA, and incubated with primary antibodies overnight at 4 °C. Thereafter, the samples were incubated with an Alexa Fluor 488-conjugated anti-mouse IgG F(ab′)2 fragment (Invitrogen, USA; 1:200), an Alexa Fluor 549-conjugated anti-rabbit IgG F(ab′)2 fragment (Invitrogen, USA; 1:200), or an Alexa Fluor 488-conjugated anti-rabbit IgG F(ab′)2 fragment (Invitrogen, USA; 1:200) at room temperature for 30 min in the dark, and were further developed by treating with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, USA; 1:300) to detect nuclei. Cells were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany), see Additional file 6 : Table S7 for antibodies used.
Alexa fluor 488 conjugated anti mouse igg f ab 2 fragment
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated anti-mouse IgG F(ab')2 fragment is a secondary antibody used for detection and visualization in immunoassays. It is composed of the F(ab')2 portion of anti-mouse IgG antibodies conjugated to the Alexa Fluor 488 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp2 confocal laser scanning microscope, by Leica (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 549 conjugated anti mouse igg f ab 2 fragment, by Thermo Fisher Scientific (2 mentions)
α sma antibody, by Abcam (2 mentions)
E cadherin antibody, by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Vimentin antibody, by Merck Group (1 mentions)
5 protocols using alexa fluor 488 conjugated anti mouse igg f ab 2 fragment
1
Immunofluorescence Staining Protocol
2
Quantification of plasmacytoid dendritic cells
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Tissue sections were deparaffinized and rehydrated with xylene, gradient ethanol, and distilled water. After rinsing thrice with PBS, the tissue sections were blocked with 3% BSA for 30 min and incubated with anti-CD123 antibody (10 µg/mL; goat; R&D Systems, USA) overnight at 4 °C. Tissue sections were then washed and incubated for 30 min with an Alexa Fluor 488-conjugated anti-mouse IgG F(ab') 2 fragment (1:200; Invitrogen, USA). Cells were co-stained with 4′,6-diamidino-2-phenylindole (1:300; Invitrogen) to detect nuclei, after which they were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany). Based on CD123 staining, pDC infiltrates were considered low if < 10 pDC per high-power field (HPF, 400×) were observed, high if > 10 pDC per HPF were observed 23 (link).
3
Immunofluorescence Staining for pDC
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Tissue sections were depara nized and rehydrated with xylene, gradient ethanol, and distilled water.
After rinsing thrice with PBS, the tissue sections were blocked with 3% BSA for 30 min and incubated with anti-BDCA-2 antibody (10 µg/mL; goat; R&D Systems, USA) overnight at 4 °C. Tissue sections were then washed and incubated for 30 min with an Alexa Fluor 488-conjugated anti-mouse IgG F(ab ) 2 fragment (1:200; Invitrogen, USA). Cells were co-stained with 4′,6-diamidino-2-phenylindole (1:300; Invitrogen) to detect nuclei, after which they were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany). Based on BDCA-2 staining, the pDC in ltrates were assessed based on the same criteria applied in IHC analysis. Based on BDCA-2 staining, pDC in ltrates were considered low if < 10 pDC per high-power eld (HPF, 400×) were observed, high if > 10 pDC per HPF were observed [22] (link).
After rinsing thrice with PBS, the tissue sections were blocked with 3% BSA for 30 min and incubated with anti-BDCA-2 antibody (10 µg/mL; goat; R&D Systems, USA) overnight at 4 °C. Tissue sections were then washed and incubated for 30 min with an Alexa Fluor 488-conjugated anti-mouse IgG F(ab ) 2 fragment (1:200; Invitrogen, USA). Cells were co-stained with 4′,6-diamidino-2-phenylindole (1:300; Invitrogen) to detect nuclei, after which they were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany). Based on BDCA-2 staining, the pDC in ltrates were assessed based on the same criteria applied in IHC analysis. Based on BDCA-2 staining, pDC in ltrates were considered low if < 10 pDC per high-power eld (HPF, 400×) were observed, high if > 10 pDC per HPF were observed [22] (link).
4
Immunofluorescence Assay for Epithelial-Mesenchymal Transition
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Cells were seeded on the cover-slips in 24-well plates and cultured overnight, then fixed in 4% paraformaldehyde for 20 min. After washing with PBS, cells were permeabilised in 0.25% Triton X-100 for 15 min, washed with PBS, and blocked with 5% BSA for 60 min. Then cells were incubated with α-SMA antibody (Abcam, USA; 1:100), E-cadherin antibody (Sigma, USA; 1:100) overnight at 4 °C. After washing with PBS, cells were incubated with an Alexa Fluor 488-conjugated anti-mouse IgG F(abʹ)2 fragment (Invitrogen, USA; 1:200) or an Alexa Fluor 549-conjugated anti-mouse IgG F(abʹ)2 fragment (Invitrogen, USA; 1:200) at room temperature in the dark. The cells were observed using a fluorescence microscope (Nikon, Tokyo, Japan).
5
Immunocytochemical Analysis of Cytoskeletal and Nuclear Proteins
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After the pretreatment as indicated, the cells grown on cover slips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After the cells were blocked in 3% BSA for 30 min, they were incubated with α-SMA antibody (Abcam, USA; 1:100), Vimentin antibody (Sigma, USA; 1:100) and p65 antibody (CST, USA; 1:800) overnight at 4°C, then washed and incubated for 30 min with an Alexa Fluor 488-conjugated anti-mouse IgG F(abʹ)2 fragment (Invitrogen, USA; 1:200) or an Alexa Fluor 549-conjugated anti-mouse IgG F(abʹ)2 fragment (Invitrogen, USA; 1:200) at room temperature in the dark. Cells were co-stained with 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen, USA; 1:300) to detect nuclei. Cells were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany).
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