Lung cancer cell line A549 was grown on coverslips to 70% confluence, then all cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.5% TritonX-100 for 10 minutes after 24 hours. Blocking was performed with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 hour at the room temperature (RT). After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). Finally, the immunofluorescence signals were visualized and recorded by Leica SP5II confocal microscope.
Alexa fluor 488 goat anti rabbit igg h c
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor®488 goat anti-rabbit IgG (H + C) is a fluorescently-labeled secondary antibody used to detect and visualize rabbit primary antibodies. It is conjugated with the Alexa Fluor®488 dye, which emits green fluorescence when excited by an appropriate light source.
Automatically generated - may contain errors
Lab products found in correlation
Antifade mounting medium, by Beyotime (7 mentions)
Albumin bovine 5, by Solarbio (4 mentions)
Bx53 microscope, by Olympus (4 mentions)
Sp5 2 confocal microscope, by Leica (3 mentions)
A8020, by Solarbio (3 mentions)
4 6 diamidino 2 phenylindole dapi c1006, by Beyotime (3 mentions)
Ab53098, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
49 6 diamidino 2 phenylindole dapi, by Beyotime (1 mentions)
Antibody against nqo1, by Cell Signaling Technology (1 mentions)
Sp5ii clsm microscope, by Leica (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride, by Beyotime (1 mentions)
Alexa fluor 568 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
8 protocols using alexa fluor 488 goat anti rabbit igg h c
1
Immunofluorescence Staining of NQO1 in A549 Cells
2
Subcellular Localization of Paip1 Protein
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IF staining was used to analyze the subcellular localization of the Paip1 protein in MGC-803 and SGC-7901 cells. The cells were grown on coverslips to 65–75% confluence, fixed with 3.65% paraformaldehyde for 5 min and penetrated with 0.5% TritonX-100 for 12 min. After blocking with 3% bovine serum albumin fraction V (A8020, Solarbio, Beijing, China) for 1.5 h, the slides were quickly washed with phosphate-buffered saline (PBS) three times. Then, the cells were incubated overnight at 4 °C with a primary antibody followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (H + C) (A11008, 1:1000 dilution, Invitrogen, USA) for 1 h. After washing with PBS, the cells were counterstained with diamidino phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and fixed on a slide. Finally, the IF signals were observed and imaged using a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.
3
Immunofluorescence Analysis of DEK and β-Tubulin
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MKN-1 gastric cancer cells grown on coverslips was fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.5% TritonX-100 for 10 min. Then washed again with PBS and blocking was performed with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for one hour at the room temperature (RT). Primary antibodies against DEK (1:50; BD Biosciences, USA) and β-Tubulin (1:50; Santa Cruz Biotechnology, USA) were incubated with cells at 4°C overnight. After more washes, cells were incubated with Alexa Fluor® 488 Goat Anti-Rabbit IgG (H + C) (A11008, 1:1000, Invitrogen, USA) and Alexa Fluor® 568 Goat Anti-Mouse IgG (H + L) (A11004, 1:1000, Invitrogen, USA) for 1 h. Subsequently, cells were washing again with PBS and counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (C1006, Beyotime, China). Coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, China) [17 (link)]. Finally, the immunofluorescence signals were visualized and recorded by Leica SP5II confocal microscope.
4
HBXIP Subcellular Localization in SKOV-3 Cells
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IF staining was used to detect the sub–cellular localization of HBXIP in SKOV-3 ovarian cancer cells. All steps were performed at RT. SKOV-3 cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min. After 24 h, cells were permeabilized with 0.5% TritonX-100 for 10 min. After blocking with 3% bovine albumin fraction V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the HBXIP antibody (1:1000) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (H + C) (A11008, 1:1000, Invitrogen, USA) for 1 h. After washing with PBS, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). IF signals were visualized and recorded with a BX53 Olympus microscope.
5
Immunofluorescence Assay of Mortalin in Breast Cancer Cells
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Breast cancer MDA-MB231 cells were grown on coverslips to 70 % confluence. The cells were fixed with 4 % paraformaldehyde for 10 min, and after 24 h cells were permeabilized with 0.5 % TritonX-100 for 10 min. Blocking was performed with 3 % Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h at room temperature. After washing with PBS, cells were incubated with rabbit anti-Mortalin antibody (ab53098) (Abcam, Cambridge, MA, USA), at 4 °C overnight, followed by incubation with Alexa Fluor 488 Goat Anti-Rabbit IgG (H + C) (A11008, 1:1000, Invitrogen, USA) for 1 h at room temperature. After washing with PBS, the cells were counterstained with DAPI (C1006, Beyotime, Shanghai, China), and the coverslips were mounted with an Anti fade Mounting Medium (P0126, Beyotime, Shanghai, China). IF signals were visualized and recorded with a BX53 Olympus microscope.
6
Immunofluorescence Assay of Tiam1 in MDA-MB-231 Cells
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The breast cancer cell line, MDA-MB-231, was grown on coverslips to 70 % confluence and then the cells were fixed in 4 % paraformaldehyde in PBS for 10 min at RT, and permeabilized with 0.5 % TritonX-100 for 10 min. Blocking was performed with 3 % bovine serum albumin fraction V (A8020, Solarbio, Beijing, China) for 1 h at RT. After washing with phosphate-buffered saline (PBS), cells were incubated with anti-rabbit Tiam1 (1:500, Santa Cruz Biotechnology) at 4 °C overnight, followed by incubation with Alexa Fluor® 488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 h at RT. After washing with PBS, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). Finally, the immunofluorescence signals were visualized and recorded by Leica SP5II confocal microscope.
7
Subcellular Localization of HBXIP in NSCLC
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An immunofluorescence (IF) staining method was used to detect the subcellular localization of the HBXIP protein in A549 NSCLC cells. All steps were performed at RT. A549 cells were grown on coverslips to reach 70%-80% confluence and fixed with 4% paraformaldehyde for 10 min; after 24 h, the cells were permeabilized with 0.5% Triton X-100 for 10 min. After blocking with 3% Albumin Bovine V (A8020; Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the HBXIP antibody (1:1000) at 4°C overnight, followed by incubation with Alexa Fluor 488 Goat Anti-Rabbit IgG (H + C; A11008, 1:1000; Invitrogen) for 1 h. After washing with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; C1006; Beyotime, Shanghai, China), and the coverslips were mounted with Antifade Mounting Medium (P0126; Beyotime). Finally, the IF signals were visualized and recorded using a BX53 Olympus microscope.
8
Mortalin Immunofluorescence in A549 NSCLC Cells
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The A549 NSCLC cell line was grown on coverslips to 70%-80% confluence, and then all of the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Subsequently, after blocking with 3% bovine albumin fraction V (A8020; Solarbio, Beijing, China) for 1 h and washing with phosphate-buffered saline (PBS), the cells were incubated with rabbit anti-Mortalin antibody (ab53098; Abcam, Cambridge, MA, USA), at 4°C overnight and then incubated with Alexa Fluor ® 488 Goat Anti-Rabbit IgG (H+C) (A11008, 1:1000; Invitrogen, CA, USA) for 1 h. Next, the cells were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI; C1006, Beyotime, Shanghai, China). The coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). Finally, the immunofluorescence (IF) signals were visualized and recorded using a BX53 Olympus microscope.
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