LX2 cells were grown on glass coverslips and treated as described in the Results and figure legends. Then, cells were washed twice with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min and processed for immunofluorescence with the indicated primary antibodies: anti-αSMA (A-2547) purchased from Sigma-Aldrich, anti-COL1A1 (sc-8784) and anti-MMP9 (sc-6840) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the appropriate FITC-conjugated secondary antibodies [Alexa Fluor® 488 goat anti-rabbit IgG (A11034) or Alexa Fluor® 488 goat anti-mouse IgG (A11001), Life Technologies, Grand Island, NY, USA]. Mounting medium used was Fluoromont G® (BioNova cientifica, Madrid, Spain). Immunofluorescence was examined using a confocal microscope (Leica TCS SP5 X, Barcelona, Spain) and image analysis procedures were performed with Fiji software (NIH, Bethesda, MD).
Alexa fluor488 goat anti mouse igg a 11001
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor488 Goat Anti-Mouse IgG (A-11001) is a fluorescently labeled secondary antibody. It is designed to bind to mouse IgG antibodies and can be used for detection and visualization applications in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 a 11008, by Thermo Fisher Scientific (2 mentions)
594 a 11012 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bz 9000 microscope, by Keyence (2 mentions)
Vectashield mounting medium with dapi h 1200, by Vector Laboratories (2 mentions)
Alexa fluor 488 goat anti rabbit igg a11034, by Thermo Fisher Scientific (2 mentions)
Anti α sma a2547, by Merck Group (1 mentions)
Tcs sp5x, by Leica (1 mentions)
Cd68 d4b9c, by Cell Signaling Technology (1 mentions)
Sybr green mix, by Takara Bio (1 mentions)
6 protocols using alexa fluor488 goat anti mouse igg a 11001
1
Immunofluorescence of Activated Hepatic Stellate Cells
2
Immunocytochemistry of Pluripotent Stem Cells
3
Immunofluorescent detection of viral antigen
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The medium of infected cells was removed and the cells were washed once with phosphate buffered saline (PBS). Subsequently, the cells were fixed using 100% ice-cold methanol (10 minutes at -20°C). To remove residual methanol, the cells were washed twice with PBS. Staining with the primary antibody was carried out for 20 minutes at room temperature (RT) with an anti-immediate-early antigen antibody (anti-I.E.A., 11–003, Argene) diluted 1/100 in PBST (PBS containing 0.05% tween-20). Prior to incubation with anti-mouse antibody conjugated to Alexa Fluor 488 (Alexa Fluor 488 goat anti-mouse IgG, A-11001, Life Technologies), the cells were washed twice with PBS. After 20 minutes incubation at RT, the secondary antibody was removed and the cells were washed twice with PBS. Afterwards, the cells were stored at 4°C in 200μl PBS until readout.
4
Immunocytochemistry of Pluripotent Stem Cells
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iPS cells cultured in 24-well plates were washed with 500
μl/well PBS, fixed with 250 μl/well 4% paraformaldehyde
at room temperature for 20 minutes, and then washed with 500 μl/well PBS
three times. The cells were blocked and permeabilized with 250 μl/well
PBS with 0.1% Triton-X (PBS-T) supplemented with 5% bovine serum
albumin (BSA) at room temperature for 1 hour, and then in 200 μl/well
primary antibodies diluted 200 times in PBS-T with 5% BSA at 4°C
overnight. The primary antibodies (all purchased from Abcam) used in this study
were SOX2 (ab59776), OCT4 (ab19857), SSEA4 (ab16287), and TRA-1-81 (ab16289).
The cells were washed with 500 μl/well PBS-T three times, incubated with
200 μl/well secondary antibodies diluted 500 times in PBS-T with
5% BSA at room temperature for 1 hour, and then washed with 500
μl/well PBS three times. The secondary antibodies used were Alexa Fluor
488 Goat Anti-Mouse IgG (A-11001) and Alexa Fluor 488 (A-11008) or 594 (A-11012)
Goat Anti-Rabbit IgG (Life Technologies). Finally, the cells were mounted with
75 μl/well VECTASHIELD Mounting Medium with DAPI (H-1200) (Vector
Laboratories). The pictures were taken by using BZ-9000 microscope
(Keyence).
μl/well PBS, fixed with 250 μl/well 4% paraformaldehyde
at room temperature for 20 minutes, and then washed with 500 μl/well PBS
three times. The cells were blocked and permeabilized with 250 μl/well
PBS with 0.1% Triton-X (PBS-T) supplemented with 5% bovine serum
albumin (BSA) at room temperature for 1 hour, and then in 200 μl/well
primary antibodies diluted 200 times in PBS-T with 5% BSA at 4°C
overnight. The primary antibodies (all purchased from Abcam) used in this study
were SOX2 (ab59776), OCT4 (ab19857), SSEA4 (ab16287), and TRA-1-81 (ab16289).
The cells were washed with 500 μl/well PBS-T three times, incubated with
200 μl/well secondary antibodies diluted 500 times in PBS-T with
5% BSA at room temperature for 1 hour, and then washed with 500
μl/well PBS three times. The secondary antibodies used were Alexa Fluor
488 Goat Anti-Mouse IgG (A-11001) and Alexa Fluor 488 (A-11008) or 594 (A-11012)
Goat Anti-Rabbit IgG (Life Technologies). Finally, the cells were mounted with
75 μl/well VECTASHIELD Mounting Medium with DAPI (H-1200) (Vector
Laboratories). The pictures were taken by using BZ-9000 microscope
(Keyence).
5
Immunofluorescence Staining of Neuronal Markers
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Antibodies against the neurofilament (NF, C28E10) and synapsin (SYN, D12G5) were purchased from Cell Signaling Technology. The antibody against S100β (IR504) was purchased from Dako. CF568 α-bungarotoxin (#00006, 1:3000 for staining) was purchased from Biotium. CD68 (D4B9C) was purchased from Cell Signaling. AlexaFluor-488 goat anti-rabbit IgG (A-11034) and AlexaFluor-488 goat anti-mouse IgG (A-11001) were purchased from Invitrogen. β-galactosidase (β-Gal, SAB3500043) was purchased from Sigma. The remaining chemicals were purchased from Sigma-Aldrich unless otherwise stated. SYBR green mix (100029284) was purchased from Takara. A high-capacity cDNA Reverse Transcription kit (4368814) was purchased from ABI. In addition, μ-conotoxin GIIIB (H-9015.1000BA) was purchased from Bachem Americas.
6
Acetylated Tubulin Staining for Primary Cilia
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Staining was performed according to Alsolami et al (2019) (link). Cells were seeded on chamber slides and grown to 80% confluency before serum starving in DMEM with 0.5% FBS for 48 h. Media were removed, and cells were washed with 1× PBS, fixed in cold (−20°C) methanol for 10 min, and washed once more in 1× PBS for 5 min. Cells were blocked in 1× PBS with 0.2% Triton X-100 (PBX) at 37°C for 5 min and then in PBX with 3% BSA for 30 min at 37°C. Slides were moved to a humidified chamber with primary antibody (anti-acetyl-alpha-tubulin, clone 6-11B-1, MABT868; Sigma-Aldrich) diluted in PBX (1:250) added for 1 h at room temp. Slides were washed for 3 × 5 minutes in PBS and placed back in a covered humidified chamber. The secondary antibody (Alexa Fluor 488 goat anti-mouse IgG, A11001; Invitrogen) was diluted in PBX (1:500) and added to coverslips for 1 h at room temp. Slides were washed for 3 × 5 min in PBS, mounted with Fluoromount-G, and imaged with 60× and 100× oil immersion objectives on the spinning disk confocal microscope (Alsolami et al, 2019 (link)). Maximum-intensity projections were created in FIJI for each image, and primary cilia were measured using the segmented line tool for the acetylated tubulin staining.
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