Human colon biopsies and mice bowel samples were embedded into Shandon cryomatrix (Thermo Fisher Scientific) and cut into 5 μm slides, stored at -80 °C until use. HT-29 and CCD-18Co cells were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD) for 15 min at RT, then incubated with primary antibodies specific to αSMA (sc-53015; mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at RT. After repeated washing slides were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen) and Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen), both diluted to 1:100 for 30 min at RT in the dark and counterstained with Hoechst 33,342 (1:2000, Sigma-Aldrich). Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).
Alexa fluor 568 donkey anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
The Alexa Fluor 568 donkey anti-rabbit secondary antibody is a fluorescently labeled antibody that binds to rabbit primary antibodies. It can be used in various immunoassay techniques, such as immunofluorescence microscopy, to detect and visualize target proteins or antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Panoramic 250 flash 3, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mice monoclonal ab16999, by Abcam (2 mentions)
Alexa fluor 488 donkey anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab124332, by Abcam (1 mentions)
Vectashield fluorescent mounting medium, by Vector Laboratories (1 mentions)
Sc 53015, by Santa Cruz Biotechnology (1 mentions)
Shandon cryomatrix, by Thermo Fisher Scientific (1 mentions)
C2 confocal laser scanning microscope system, by Nikon (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Facs permeabilizing solution 2, by BD (1 mentions)
Facsaria cytometer, by BD (1 mentions)
Triton, by Avantor (1 mentions)
Alexa fluor 568 donkey anti goat secondary antibody, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rabbit anti jnk, by Cell Signaling Technology (1 mentions)
Rabbit anti p jnk, by Cell Signaling Technology (1 mentions)
Anti mouse rabbit hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
7 protocols using alexa fluor 568 donkey anti rabbit secondary antibody
1
Immunofluorescence Analysis of Intestinal Tissues
2
Histological Analysis of Tumor and Adipose
3
Histological Analysis of Tumor and Adipose
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Tumors and mammary glands (in obese tumor adjacent and in lean normal 4th gland contralateral to the injected tumor-bearing mammary glands) were isolated at the time of sacrifice and fixed in 10% formalin. Then stored in 70% ethanol at room temperature before embedding. Formalin-fixed paraffin embedded (FFPE) sections from tumors and adipose were cut at 5 μm thickness. FFPE sections were stained with Hematoxylin and Eosin, for mitosis and adipocyte size, and scanned by Thermo Fisher digital scanner (Panoramic 250 Flash III, Thermo Fisher, Tewksbury, MA) and quantified using software (Case Viewer). Mitotic nuclei were scored by pathologist (R. Sekhri) by examining 10–20 40X high power fields (HPF) for mitotic nuclei to calculate the mitotic index (number of mitosis/10 HPF). Tumor sections from obese were deparaffinized and rehydrated. Sections were then incubated with the primary antibody against anti-CD3 antibody (Rabbit anti-mice monoclonal ab16999 Abcam) and Alexa fluor 568 donkey anti-rabbit secondary antibody (Invitrogen) was used to detect the primary antibody. Images were captured using Zeiss 710 with 20 × numerical aperture objective lens. Total number of CD3+ cells were quantified using ImageJ 1.8 (Rueden et al., 2017 (link)).
4
Intracellular Cytokine Profiling in HT-29 Cells
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HT-29 cells were centrifuged, washed with PBS and incubated for 10 min at RT with FACS Permeabilizing Solution 2 (BD). Permeabilized cells were washed with PBS and incubated with primary anti TGF-ß1 (sc-146, rabbit, Santa Cruz) or anti PDGF-B (sc-7878, rabbit, Santa Cruz) antibody diluted to 1:50 for 30 min at RT. Cells were subsequently washed with Permeabilizing Solution 2 and incubated with Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen) diluted to 1:200 for 30 min at RT in the dark. Negative controls were incubated with the secondary antibody alone. The flow cytometric analysis was carried out using a FACSAria cytometer (BD). The mean fluorescence intensity (MFI) values of each sample were normalized and presented as the ratio of their control values.
5
Visualizing Endothelial Cell Junctions
6
Protein Expression and Oxidative Stress
7
Immunostaining of autophagy proteins
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Following the treatment protocol, cells were fixed using a 1:1 mixture of pre-warmed DMEM and 8% paraformaldehyde. Cells were rinsed 3 times using 1 × PBS and permeabilised using 0.1% Triton X-100 for 6 min. Cells were subsequently washed three times using 1 × PBS, blocked in 3% BSA for 30 min and incubated in either p62 or NBR1 primary antibody at 4 °C overnight. Next, cells were washed with 1 × PBS before adding Alexa Fluor 568 donkey anti-rabbit secondary antibody (#A10037, Life Technologies) for 2 h at 4 °C. Primary and secondary antibodies were made up in a 1:200 dilution using 1 × PBS. Cells were then washed with 1 × PBS and samples mounted using fluorescence mounting medium (Dako, Santa Clara, CA, USA).
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