Briefly, 4‐μm‐thick murine lung tissue slices were dewaxed and rehydrated in a graded series of ethanol. Next, their endogenous peroxidase activity was blocked and the sections were subjected to epitope retrieval, following which, they were incubated with primary antibodies against Anti‐mouse CD3e‐biotin (dilution 1:100 eBio500A2, eBioscience,Cal, USA), Anti‐rat GATA3 (dilution 1:50, ab110093, Abcam, Mass, USA), and Anti‐rabbit inducible T‐cell costimulatory (ICOS) (dilution 1:200, ab175401, Abcam, Mass, USA). Bound biotinylated antibodies were detected using streptavidin‐AF555 conjugated secondary antibodies (S32355, Invitrogen, Carlsbad, Cal, USA), then anti‐GATA3 antibodies were detected using rabbit anti‐rat IgG‐FITC‐AF488 (A‐11006, Invitrogen, Carlsbad, Cal, USA). Finally, ICOS antibodies were detected using goat anti‐rabbit IgG‐647 secondary antibodies (A‐27040, Invitrogen, Carlsbad, Cal, USA). Sections were counterstained with 4’,6‐Diamidino‐2’‐phenylindole (62248, Invitrogen, Carlsbad, Cal, USA). Images were acquired under an Laser Scanning Confocal Microscope (LSM) 510 meta confocal microscope (Zeiss, Oberkochen,UK) and the Zeiss LSM software was used for image analysis.
Ab175401
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab175401 is an immunoaffinity resin designed for the purification of target proteins from cell lysates or other biological samples. This resin utilizes a proprietary ligand that binds to the target protein with high specificity and affinity, allowing for efficient capture and recovery of the desired protein.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Ebio500a2, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Inverted fluorescence microscope, by Zeiss (1 mentions)
Optimum cutting temperature compound, by Sakura Finetek (1 mentions)
Ab62516, by Abcam (1 mentions)
R d hrp dab staining kit, by R&D Systems (1 mentions)
4 protocols using ab175401
1
Multicolor Immunofluorescence Imaging of Murine Lung Tissue
2
Immunohistochemical Analysis of ICOS in Rat Aorta
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Rat aortas were fixed with 4% PFA, embedded in paraffin, and cut into 4 µm slices. The tissue sections were deparaffinized and rinsed in water. After antigen retrieval and blocking in BSA solution, sections were incubated with the primary anti-ICOS antibody (Abcam, ab175401) then the secondary antibody (Alexa Fluor® 488-conjugated polyclonal goat anti-Rabbit IgG-H&L; Abcam, ab150077). Control slides were prepared according to the same procedure but without the primary antibody. The nuclei were counterstained with DAPI. Sections were mounted with a medium containing an anti-fluorescence quencher, and images were captured with a confocal microscope (LSM800, Zeiss, Oberkochen, Germany).
3
Immunofluorescence Staining for ICOS and ICOSL
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The clinical specimens were fixed by 4% PFA solution at 4 C for 48 hours and then embedded with an optimum cutting temperature compound (Sakura Finetek Japan, Nagano, Japan). Cryostat sections of 5mm were made and stored at 220 C before staining. Fluorescence immunostaining was performed as previously described 20 . The ICOS antibody (1:100, ab175401; Abcam, Cambridge, UK) or ICOS ligand antibody (1:100, ab124972, Abcam) was used as the primary antibody followed by antibody conjugated with Alexa Fluor 594 (red) (1:500, EM35153-01; EMAR; Emarbio, Beijing, China) or Alexa Fluor 488 (green) (1:500, EM35141-01, EMAR), respectively. Normal rabbit immunoglobulin G antibody (Cayman Chemical, Ann Arbor, MI) served as the negative control (thus replacing the primary antibody). The images were captured by an inverted fluorescence microscope (Zeiss) under !400 magnification.
4
Spatial Expression of ICOS, ICOS-L, RANKL
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Immunohistochemistry (IHC) of ICOS, ICOS ligand, and RANKL was performed to visualize the spatial expression of these proteins in paraffin-embedded sections from rat samples.
For IHC, all sections were stained using the R&D HRP-DAB staining kit (CTS017; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The primary antibodies were as follows: ICOS antibody (1:100, ab175401, Abcam), ICOS-ligand antibody (1:100, A7080; ABclonal, Woburn, MA), and RANKL antibody (1:500, ab62516, Abcam). The counterstaining was performed with hematoxylin. The negative controls were treated with phosphate-buffered saline rather than the primary antibodies.
For IHC, all sections were stained using the R&D HRP-DAB staining kit (CTS017; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The primary antibodies were as follows: ICOS antibody (1:100, ab175401, Abcam), ICOS-ligand antibody (1:100, A7080; ABclonal, Woburn, MA), and RANKL antibody (1:500, ab62516, Abcam). The counterstaining was performed with hematoxylin. The negative controls were treated with phosphate-buffered saline rather than the primary antibodies.
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