AMSC-Exo were labeled with a PKH26 Red Fluorescent dye (Sigma-Aldrich), at a dilution of 1:200, according to the manufacturer's instructions. Then the red dye-labeled exosomes were injected into mice via tail vein and PBS was used as controls; 6–8 h later, the animals were sacrificed and liver tissues were isolated. The liver sample was then embedded in Tissue-Tek™ CRYO-OCT (Fisher Scientific) and processed for 5 μm sections. The KCs in these sections were stained using a green dye Alexa Fluor 488-labeled CD68 (Abcam). Hepatic distribution of the PKH26-labeled AMSC-Exo and their colocalization with KCs were imaged via confocal microscopy (Olympus, Center Valley, PA, USA).
Tissue tek cryo oct
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tissue-Tek™ CRYO-OCT is a laboratory instrument designed for quick-freezing and embedding of tissue samples for cryosectioning. It provides a controlled and consistent freezing environment to prepare samples for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Pkh26 red fluorescent dye, by Merck Group (1 mentions)
Confocal microscopy, by Olympus (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Optical fractionator probe, by MBF Biosciences (1 mentions)
Axiocam, by Zeiss (1 mentions)
Lamp 1 antibody, by Abcam (1 mentions)
Dylight 488 labeled secondary antibodies, by MultiSciences Biotech (1 mentions)
Ix83 fv3000 confocal microscope, by Olympus (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Ix83 fv3000, by Olympus (1 mentions)
Smz18, by Nikon (1 mentions)
8 protocols using tissue tek cryo oct
1
Tracking Exosome Biodistribution in Liver
2
Hepatic Triglyceride Accumulation Assessment
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Hepatic TG accumulation was determined by both histochemical detection (Oil Red O staining) and biochemical assays. For Oil Red O Staining, liver tissues were frozen in Tissue-Tek CRYO-OCT (Fisher Scientific) and cryostat tissue sections were cut at 7 µm thicknesses, fixed with 10% formalin for 10 min, and stained following an Oil Red O procedure. For biochemical assays, total liver lipids were extracted using chloroform/methanol (2∶1, v/v). The protein levels in the homogenate were assayed using protein assay reagent (Bio-Rad, Hercules, CA) to normalize the amount of lipid extracted. TG levels were measured using assay kits from BioVision.
3
Quantitative Assessment of Neurodegeneration
4
Histological Evaluation of Colon Inflammation
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Cryosections (5 μm) were prepared from Cryo-OCT (Tissue-Tek; Fisher Scientific, Schwerte, Germany) embedded colon tissue. After the slides had been fixed for 10 minutes, we stained them with H&E. The sections were independently evaluated by two blinded investigators using a scoring system to grade inflammation and cell infiltration from 0 to 3 (0 = none; 1 = mild; 2 = moderate; 3 = severe) and the occurrence of ulcerations from 0 to 3 (0 = none; 1 = ulcers spanning up to two crypts; 2 = ulcers spanning 3 to 10 crypts; 3 = ulcers spanning more than 10 crypts). The grading was performed for the proximal and the distal part of the colon separately, with a combined maximal grading score of 12. For the staining of infiltrating phagocytes, a monoclonal antibody raised against CD11b (clone: M1/70; BD Biosciences) was used, and the presence of regulatory T cells was evaluated using a monoclonal antibody against Foxp3 (clone: MF-14; Biolegend). Secondary antibody binding and subsequent peroxidase reaction was performed as described previously.20 (link)
5
Imaging EGFP Expression in Buffalo Ear Tissue
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Buffalo ear tissue was harvested by removing the hairs with a scalpel blade, rinsing thoroughly in cold PBS, and fixing the cells in 4 % fresh paraformaldehyde at 4 °C overnight. The tissues were washed and perfused in gradient concentration of sucrose solution (5 %, 10 %, 15 %, 30 %) at 4 °C, before embedding in CRYO-OCT Tissue-Tek™ (Fisher Scientific, USA). Cryo-sections were cut at 15 μm thickness and observed under a confocal laser scanning microscope (Zeiss LSM 510META, Germany) to identify EGFP expression. Images were acquired with an AxioCam (Zeiss), keeping all microscope and laser settings kept constant between different groups and replicates. Brightfield and fluorescent images were digitally enhanced for brightness and contrast in Corel Paint Shop Pro XI (‘Histogram adjustment’). The same settings were used for images of all three groups.
6
Lysosome Inhibition Impacts PD-L1 Trafficking
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The murine PMs were cultured overnight on glass coverslips and then treated with lysosome inhibitors for 24 h. Then, the cells were coincubated with labeled αPD-L1 or TEV-bound αPD-L1 for another 2 h. After being washed three times with PBS, the cells were fixed with precooled methyl alcohol for 10 min at −20 °C and then permeabilized with 0.1% Triton X-100 for 10 min at RT. After the cells were blocked with 5% BSA and 3% goat serum in PBS, they were incubated with LAMP1 antibodies (Abcam, Cambridge, UK) overnight at 4 °C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech, Hangzhou, China) for 30 min at RT and washed in PBS. Finally, nuclei were stained with DAPI (Thermo Fisher Scientific). Liver and spleen tissues were embedded in Tissue-Tek™ CRYO-O.C.T. (Thermo Fisher Scientific) and processed to obtain 5 μm sections. Then, the tissue sections were stained with mouse F4/80 antibodies (Abcam) at 4 °C overnight followed by staining with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech) for 1 h at 4 °C. The nuclei were stained with DAPI (Thermo Fisher Scientific) for 20 min at RT. The stained sections were imaged using an Olympus IX83-FV3000 confocal microscope (Olympus). Images were analyzed with ImageJ software (NIH).
7
Immunofluorescent Staining of Tissue Sections
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Tissues were embedded in Tissue‐Tek Cryo‐O.C.T (Thermo Fisher Scientific) and processed to obtain 5 μm sections. Then, the tissue sections were stained with primary antibodies at 4°C overnight, followed by staining with the corresponding fluorescence‐labelled secondary antibodies at 4°C for 1 h. Finally, the nuclei were stained with 0.5 μg/ml DAPI for 20 min at RT. The stained sections were observed with a Nikon A1R confocal microscope (Nikon). The CD31+ cell area was analysed by the vessel analysis plugin in ImageJ (NIH, Bethesda, MD, USA). The percent of CD31+ cell area was determined and normalized to the area of the total cells. The antibodies used and the corresponding dilutions are listed in Table S2 .
8
Visualizing Tumor-Derived Extracellular Vesicle Uptake and Distribution
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To detect the uptake of TEVs by MCs, p-pMCs and 40L cells were treated with 2.5 μg ml-1 CFSE-labeled LLC-EVs for 24 h. To detect the distribution of TEVs in vivo, 100 μg of VivoTrack 680-labeled LLC-EVs were intravenously or intrapleurally injected into mice. The mice were euthanized 24 h later, and the brain, heart, lungs, liver, spleen, kidneys and organs of the gastrointestinal tract were collected, and images were taken under an IVIS (PerkinElmer). To detect the distribution of TEVs in the lungs, 20 μg of PKH26-labeled LLC-EVs were intravenously or intrapleurally injected into mice. The lungs of these mice were collected 24 h later, embedded in Tissue-Tek™ Cryo-O.C.T. Compound (Thermo Fisher Scientific) and processed to obtain 10 μm sections. Nuclei were stained with 0.5 μg ml-1 DAPI for 20 min at RT. The cells and stained sections were observed by confocal microscopy (Olympus IX83-FV3000). To detect pleural TEV uptake, 20 μg of CFSE-labeled LLC-EVs were intrapleurally injected into mice. In some experiments, 0.25 mg kg-1 Cyto-D was administered by intrapleural injection 2 h before EV injection. The pleura of these mice was collected 24 h later and observed by stereomicroscopy (Nikon SMZ18, Tokyo, Japan).
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