Mice were injected in the footpad with 30 μl of PBS containing 107 colony-forming units (CFU) P. aeruginosa48 (link). Four hours post infection the draining popliteal LNs were harvested and fixed with PLP buffer (0.05 M phosphate buffer containing 0.1 M L-lysine [pH 7.4], 2 mg/ml NaIO4, and 10 mg/ml paraformaldehyde) for 12 h, then dehydrated in 30% sucrose prior to embedding in OCT freezing media (Sakura Finetek). 30 μm sections were cut on a CM3050S cryostat (Leica), adhered to Superfrost Plus slides (VWR), stained, mounted with Fluormount G (Southern Biotech), and imaged on a LSM 710 confocal microscope (Carl Zeiss Microimaging).
Oct freezing media
Manufactured by Sakura Finetek
Sourced in Japan
OCT freezing media is a specialized liquid used for cryopreservation of tissue samples for further analysis using optical coherence tomography (OCT) imaging techniques. It is designed to provide optimal preservation of tissue morphology and integrity during the freezing process.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Superfrost plus slides, by Avantor (2 mentions)
Fluormount g, by Southern Biotech (2 mentions)
Cm3050s cryostat, by Leica camera (2 mentions)
Streptavidin biotin blocking kit, by Vector Laboratories (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Antigenfix, by Diapath (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Cm1850 cryostat, by Leica Microsystems (1 mentions)
4 protocols using oct freezing media
1
Visualizing Lymph Node Infection
2
Visualizing Lymph Node Infection
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Mice were injected in the footpad with 30 μl of PBS containing 107 colony-forming units (CFU) P. aeruginosa48 (link). Four hours post infection the draining popliteal LNs were harvested and fixed with PLP buffer (0.05 M phosphate buffer containing 0.1 M L-lysine [pH 7.4], 2 mg/ml NaIO4, and 10 mg/ml paraformaldehyde) for 12 h, then dehydrated in 30% sucrose prior to embedding in OCT freezing media (Sakura Finetek). 30 μm sections were cut on a CM3050S cryostat (Leica), adhered to Superfrost Plus slides (VWR), stained, mounted with Fluormount G (Southern Biotech), and imaged on a LSM 710 confocal microscope (Carl Zeiss Microimaging).
3
Visualization of Splenic MCMV Infection
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Spleen fragments isolated from uninfected or MCMV‐infected IFNbEYFP reporter mice were fixed for 2 h with Antigenfix (DiaPath), then washed in phosphate buffer (PB1X: 0.025 M NaH2PO4 and 0.1 M Na2HPO4) for 1 h, dehydrated in 30% sucrose overnight at 4°C, and embedded in OCT freezing media (Sakura Finetek). Eight‐micrometer‐thick tissue sections were blocked in PB1X containing 0.1% saponin, 2% BSA, 2% 2.4G2 supernatant, and streptavidin/biotin blocking kit (Vector Laboratories) and stained in PB1X with the following antibodies: Alexa488‐conjugated rabbit anti‐GFP (Invitrogen), mouse Croma anti‐MCMV IE‐1 mAb, followed by Alexa633‐conjugated goat anti‐mouse IgG2a mAb (Molecular Probes) and biotin rat anti‐mouse CD169 (MOMA1, Abcam), followed by Alexa546‐conjugated streptavidin (Thermo Fisher). Stained sections were mounted in ProLong Gold Antifade reagent (Invitrogen), acquired on a ZEISS LSM780 confocal microscope (Zeiss), and analyzed with ImageJ software.
4
Immunohistochemical Analysis of IL-33 in Lung Tissues
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Lung tissues and primary tumors were fixed in 4% paraformaldehyde for 4 h and then embedded in OCT freezing media (Sakura Finetek, Tokyo, Japan). Subsequently, 8 μm sections were cut on a CM1850 cryostat (Leica Microsystems, Nussloch, Germany) and affixed to microscope slide glasses (Matsunami, Japan). Frozen sections were blocked with 0.1% Triton-X100 (Sigma), 1% BSA and 10% FBS containing 1× PBS solution at room temperature for 1 h. After washing with 1× PBS three times, the sections were stained with rat anti-mouse IL-33 monoclonal antibody (396118, Thermo Fisher) at 4 °C overnight. To detect the signal, goat Cy3-conjugated anti-rat immunoglobulin antibody (405408, BioLegend) was used. DAPI-stained slides were examined with a BZ-X 710 microscope (Keyence, Osaka, Japan) and analyzed using ImageJ software (imagej.nih.gov/ij/ accessed on 22 April 2022).
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