Immunofluorescence was performed on cells grown on coverslips and in matrigel. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min and permeabilized with 0.5% Triton X-100 for 30 min for cells grown on coverslips or 0.2% Triton X-100 for 10 min for cells grown in matrigel. After blocking in 5% goat serum, cells were probed with each antibody as follows: anti-mouse PAX2 (1:500, Santa Cruz), anti-mouse PAX8 (1:200, Santa Cruz), anti-rabbit E-cadherin (1:200, Abcam) and anti-rat CD44 (1:200, Abcam). Alexa Fluor goat anti-mouse IgG (1:1000), goat anti-rabbit IgG (1:500) and goat anti-rat (1:500; all from Molecular Probes, Carlsbad, CA) were used as secondary antibodies. The cells on coverslips were then mounted to microscope slides using Vectashield hard set mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and the immunofluorescence images were visualized and analyzed using an inverted fluorescence microscope (Axioskop 2 MOT plus, Zeiss) and Axiovision software.
Alexa fluor goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Alexa Fluor goat anti-mouse IgG is a fluorescently labeled secondary antibody. It is designed to detect and bind to mouse immunoglobulin G (IgG) for use in various immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor goat anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Lsm 880 airyscan, by Zeiss (4 mentions)
Matrigel, by STEMCELL (2 mentions)
Matrigel, by BD (2 mentions)
Sc 398144, by Cell Signaling Technology (2 mentions)
Duo link in situ fluorescence kits, by Merck Group (2 mentions)
Inverted laser scanning lsm880 airyscan, by Zeiss (2 mentions)
Anti mouse pax2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti e cadherin, by Abcam (1 mentions)
Anti rat cd44, by Abcam (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 mot plus, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Vectashield hardset mounting medium with dapi, by Vector Laboratories (1 mentions)
Tcs sp5, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Ab6992, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
Fluoromount mounting medium, by Southern Biotech (1 mentions)
10 protocols using alexa fluor goat anti mouse igg
1
Immunofluorescence Characterization of Cells
2
Immunofluorescence Analysis of Tight Junctions
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Tissues were fixed in 4% paraformaldehyde for 48 h, treated according to standard histological tissue processing, and embedded in paraffin. For immunostaining, paraffin was removed from the cross-sections (8 μm) using a xylol–ethanol gradient. For antigen retrieval, sections were boiled in 1 mM EDTA buffer solution. To block non-specific binding sites, the sections were incubated in PBS containing 6% (vol/vol) goat serum and 1% BSA (blocking solution) for 60 min at room temperature. All subsequent washing procedures were performed with this blocking solution. Tissues were incubated at room temperature for 60 min in rabbit anti-claudin-1, -2, -3, -5, -12 (1:100), anti-occludin (1:200), and anti-tricellulin (1:500) primary antibodies in blocking solution and, after two washes, further incubated with Alexa Fluor goat anti-mouse IgG and Alexa Fluor goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) diluted to 1:500 in blocking solution for 45 min at room temperature. Furthermore, nuclei were stained with DAPI (Roche, 1:1000). Sections were mounted with ProTags MountFluor (Biocyc, Luckenwalde, Germany). Fluorescence images were obtained with a confocal laser scanning microscope (Leica TCS SP5, Wetzlar, Germany).
3
Isolation and Culture of Mouse Gastric Organoids
4
Immunofluorescence Imaging of DARPP-32, p-STAT3, and p-SRC
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Immunofluorescence was performed following cell fixation and permeabilization, using antibodies against DARPP-32 (1:400) (Santa Cruz, sc-398144), p-STAT3 (Cell Signaling, Y705, 9193s) or p-SRC (1:400) (Cell Signaling, Y416, 6943s). The cells were washed with cold PBS three times for 5 min each, and incubated with both Alexa Fluor goat anti-mouse IgG and Alexa Fluor goat anti-rabbit IgG (1:800) (Invitrogen) at room temperature for 90 min. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
5
Proximity Ligation Assay for Protein Interactions in Gastric Cancer
6
Immunofluorescence Imaging of DARPP-32, p-STAT3, and p-SRC
7
Isolation and Culture of Mouse Gastric Organoids
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The mouse stomach was cut into 2–3 mm pieces and then incubated in 5 mM EDTA for 30 minutes, with shaking at 37°C. The cell suspension passed through 40 μm cell strainers 2 times. After centrifuging at 1200 RPM for 5 minutes, the pellet was suspended in 50 μL Matrigel (BD Biosciences) and plated in pre-warmed 24-well plates. The 24-well plate was placed in a CO2 incubator for 20 minutes to allow a complete polymerization of the Matrigel, 500 μL mouse basal medium (STEMCELL, Cambridge, MA) was overlaid. Organoids were collected after 14 days, fixed in 4% paraformaldehyde for 20 min, washed in 1× PBS, and re-suspended in 30 μL Histo-gel (Richard-Allan, White Plains, NY). The histo-gel was placed into a cassette and fixed in 70% ethanol. Paraffin-embedded organoids were pretreated at 65°C for 90 min, followed by deparaffinization using standard procedures.60 (link) Antigen retrieval was performed using Tris EDTA Buffer (Genemed, Torrance, CA). Slides were then incubated for primary antibody overnight at 4°C, and further incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 2 h. Images were acquired using an inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
8
Immunostaining Protocol for Ocular Tissues
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Immunostaining was performed as our previous study 26 (link). The primary antibodies as follow: α-SMA, ab7817(the capsular bag) and ab5694 (The paraffins sections), abcam, UK; CTGF, ab6992, abcam, UK). Secondary antibodies as follow: fluorescein conjugated IgG (Alexa Fluor goat anti-mouse IgG, 1:400, Invitrogen); Strept-Avidin Biotin Complex (sp-9000, ZSGB-BIO, China).
9
Proximity Ligation Assay for Protein Interactions in Gastric Cancer
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To demonstrate the proximity (<40 nm) between two different proteins in gastric cancer cells, PLA was performed by using Duo-link In Situ-Fluorescence kits according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). The slides were mounted with the mounting solution with DAPI. Each dot represents the proximity of two interacting proteins. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
Following cell fixation and permeabilization, immunofluorescence was performed using standard methods.60 (link) The cells were washed with cold PBS three times and incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 1.5 h. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
Following cell fixation and permeabilization, immunofluorescence was performed using standard methods.60 (link) The cells were washed with cold PBS three times and incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 1.5 h. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
10
Cilia Visualization and Quantification
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The cells were washed with phosphate-buffered saline (PBS) and fixed for 10 min in 4% para-formaldehyde (PFA), followed by 3 × 5 min PBS washes, permeabilization in PBS/Triton X-100 0.2% for 10 min, 3 × 5 min PBS washes, and blocking in PBS/BSA 1% for 1 h. The cells were then incubated overnight with mouse anti-acetylated tubulin (Santa Cruz) diluted 1/500 in PBS/BSA 0.1%. On the following day, cells were washed 3 x 5 min with PBS and incubated for 1 h with secondary Alexa Fluor goat anti-mouse IgG (Invitrogen) diluted 1/300 in PBS. The slides were mounted with Fluoromount mounting medium containing DAPI (Southern Biotech) and visualized on a Nikon Eclipse fluorescent microscope (Nikon Corporation). Cilia counting was performed using ImageJ software.
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