Nasal epithelial biopsies were placed in Antigenfix (Microm Microtech) for 1–2 h at 4 °C, then 30% sucrose in PBS for 12–24 h at 4 °C, before cryopreservation in OCT (Cell Path). Sections (30 µm) were permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich), 1% normal goat serum, 1% normal donkey serum and 1% BSA (R&D) for 1–2 h at room temperature. The samples were stained with anti-human S100A9 antibodies conjugated to FITC (1 in 50 dilution, MRP 1H9, BioLegend) and anti-human EPCAM antibodies conjugated to APC (1 in 50 dilution, MRP14, BioLegend, 350703) in blocking buffer overnight and washed three times for 10 min in PBS before mounting with Fluoromount-G containing DAPI (Invitrogen). Images were acquired using a Leica SP8 confocal microscope. Raw imaging data were processed using Imaris (Bitplane).
Antigenfix
Manufactured by Microtech
Sourced in France
Antigenfix is a laboratory equipment used for the fixation and preservation of antigenic properties in biological samples. It is designed to maintain the structural and functional integrity of antigens, enabling accurate analysis and detection in various immunological and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (8 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Bovine serum albumin (bsa), by R&D Systems (2 mentions)
Fluoromount g containing dapi, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Ultrapure tris buffer, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Prolong antifade, by Thermo Fisher Scientific (1 mentions)
Cd45.2 fitc clone 104, by BD (1 mentions)
Vt1200, by Leica (1 mentions)
Type 7 a, by Merck Group (1 mentions)
Cryostat, by Leica (1 mentions)
One touch vita, by Johnson & Johnson (1 mentions)
9 protocols using antigenfix
1
Immunofluorescent staining of nasal epithelial cells
2
Immunofluorescence Staining of Nasal Epithelial Cells
3
Intravital Imaging of Skin Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trunk and ear skin pieces were fixed in Antigen Fix (Microm Microtech) for 2 h, washed in 0.1 M phosphate buffer, and dehydrated overnight in 30% sucrose in 0.1M phosphate buffer. Tissues were snap frozen, and 25-µm sections were stained with DAPI. To obtain an en face section of ear dermis, the dorsal part was split from the ventral one 2 h after intradermal ear injection of CD64-APC (2 µg), fixed in Antigen Fix for 2 h, washed in phosphate buffer, stained with DAPI, and clarified with Rapiclear 1.47 (SunJin Lab). Immunofluorescence confocal microscopy was performed by using a Zeiss LSM 880 confocal microscope. Separate images were collected for each fluorochrome and merged to obtain a multicolor image. Final image processing was performed with Imaris software (Bitplane) and Adobe Photoshop.
4
Lymph Node Staining and Imaging Protocol
5
Multicolor Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collected spleen fragments were immediately fixed with Antigenfix (Microm Microtech France, Francheville, France) for 3 hr at room temperature, followed by two washes in PBS 0•1 m pH 7•4 and a 30% sucrose bath at 4°, before embedding in OCT. Pieces were kept at -80° and sliced at 8-µm thickness. The mAbs CD45.2 (FITC clone 104; BD Pharmingen), B220 (RA3-6B2 A-647; BD Pharmingen), F4-80 (purified, clone SF12; BD Pharmingen), anti-MHC class II (M5/114-biotin; eBioscience), CD11c (N418 purified; eBioscience) Ki67 (SolA15 purifed; eBioscience) were diluted in PBS, at a pre-established concentration. Slices were incubated overnight at 4°. Secondary labelling was for 2 hr at room temperature. Prolong anti-fade (Invitrogen) containing or not DAPI was used to keep the coloured slices. Confocal microscopy was performed with the Zeiss 780 microscope with a 40× oil objective and a 0•6× zoom.
6
Cryogenic Sectioning and Immunostaining of Lymph Nodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LNs were fixed in AntigenFix (Microm Microtech) for 2 h at 4°C in the dark, washed in phosphate buffer, and dehydrated in 30% sucrose in 0.1 M phosphate buffer for 12 h. Samples were embedded in Tissue Freezing Medium (Triangle Biomedical Sciences), snap frozen, and sectioned (35 µm) on a cryostat (Leica Microsystems). Immunostaining was realized in 0.1 M UltraPure Tris Buffer (Thermo Fisher Scientific) containing 0.5% BSA and 1% Triton X-100. Confocal imaging was performed with a Zeiss LSM 880 confocal microscope. Separate images were collected for each fluorochrome and overlaid to obtain a multicolor image. Final image processing and 3D reconstruction were performed with Imaris software (Bitplane). For unfixed LN slice preparation, fresh LNs were embedded in 4% low-melting-temperature agarose (type VII-A; Sigma-Aldrich), sectioned in 300-µm-thick slices using a vibratome (Leica VT1200) in a bath of ice-cold PBS, and immediately imaged by confocal microscopy.
7
Immunofluorescence Confocal Microscopy of LNs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LNs were fixed in Antigen Fix (Microm Microtech) for 2 h, washed in 0.1 M phosphate buffer, and dehydrated overnight in 30% sucrose at 0.1 M phosphate buffer. Tissues were snap frozen and 35 µm frozen sections stained with the indicated antibodies in 0.1 M UltraPure Tris Buffer (ThermoFischer) containing 0.5% w/v BSA and 1% Triton X-100. Immunofluorescence confocal microscopy was performed using a Zeiss LSM 880 confocal microscope. Separate images were collected for each fluorochrome and overlaid to obtain a multicolor image. Final image processing was performed with Imaris software (Bitplane).
8
Epidermal Sheet Preparation for DETC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare epidermal sheets, the dorsal and ventral halves of ear skin were pulled apart as for flow cytometry and incubated in Dispase for 40 min. Epidermal sheets were separated from the dermis, fixed in AntigenFix (Microm Microtech) for 20 min at room temperature, and washed in phosphate buffer. DETCs were identified by immunostaining TCR Vγ5 expression. Immunofluorescence confocal microscopy was performed with a Zeiss LSM 780 confocal microscope. Final image processing was performed with Imaris software (Bitplane) and Adobe Photoshop.
9
Glucose and Insulin Tolerance Tests
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were placed in new cages prior to starvation. For GTTs, 12 hr fasted mice were injected intraperitoneal (i.p.) with a solution of sterile glucose (2 g/kg body weight) freshly prepared in 0.9% sterile saline. For ITTs, 6 hr fasted mice were injected i.p. with insulin diluted to 0.08 mU/μl in sterile saline for a final delivery of 0.8 mU/g body weight. Baseline glucose measurements were analyzed from tail blood before i.p. glucose or insulin injection (2 mg/g body weight) using the OneTouch Vita (LifeScan, Johnson & Johnson company) system. Blood glucose measurements were taken from the tail blood at the indicated points.
gWAT morphometry staining gWAT was fixed with Antigenfix (Microm Microtech, France), embedded in paraffin, sectioned, and stained with a hematoxylin and eosin (H&E) solution. Slides (4/group) were scanned with Axio-scan, which allowed the scanning of the entire slide at high resolution. Six pictures of six different areas from 1 to 2 sections per sample were chosen and analyzed with image analyzer software (ImageJ). Total areas of adipocytes were traced manually. The total count ranged from 3275 to 7052 adipocytes per condition. The mean surface area of the adipocytes was calculated using image analyzer software (ImageJ). For each sample, 400–1000 adipocytes were counted.
gWAT morphometry staining gWAT was fixed with Antigenfix (Microm Microtech, France), embedded in paraffin, sectioned, and stained with a hematoxylin and eosin (H&E) solution. Slides (4/group) were scanned with Axio-scan, which allowed the scanning of the entire slide at high resolution. Six pictures of six different areas from 1 to 2 sections per sample were chosen and analyzed with image analyzer software (ImageJ). Total areas of adipocytes were traced manually. The total count ranged from 3275 to 7052 adipocytes per condition. The mean surface area of the adipocytes was calculated using image analyzer software (ImageJ). For each sample, 400–1000 adipocytes were counted.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!