Orthotopic allografts were generated as in the efficacy studies. When tumors reached 200–400 mm3 (about 2 weeks post-cell injection), R848-Toco/HA-Toco (25 μg on R848 basis) in 50 μL volume was injected intratumorally. After 24 h, tumors were dissected and embedded in OCT medium (Fisher Scientific), followed by storage at −80 °C. Tumors were sectioned (10 μm) with a cryotome. Sections were fixed for 2 × 10 min with acetone and washed with PBS. Primary antibodies were diluted to 5 μg/mL in blocking buffer (5% goat serum in PBS) and incubated overnight at 4 °C. Antibodies used were Alexa Fluor® 488 anti-CD8a, Alexa Fluor® 594 anti-CD11b, and Alexa Fluor® 647 anti-CD11c (BioLegend). After antibody staining, sections were stained with DAPI (0.5 μg/mL in PBS; Invitrogen) for 10 minutes, mounted in SouthernBiotech™ Fluoromount-G™ Slide Mounting Medium and stored in the dark at 4 °C. Images were acquired using an Olympus IX-81 inverted epifluorescence microscope at 10x magnification. and images were captured with a Hamamatsu EM-CDD Digital Camera. The entire tumor section was imaged. Many 10x images were montaged together using SlideBook 6 to view the whole section.
Fluoromount g slide mounting medium
Manufactured by Southern Biotech
Sourced in United States
Fluoromount-G™ Slide Mounting Medium is a non-hardening, aqueous-based mounting medium designed for use with fluorescent-labeled specimens. It is formulated to preserve the fluorescent signal and provide a clear, refractive index-matched mounting environment for microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 anti cd8a, by BioLegend (1 mentions)
Alexa fluor 594 anti cd11b, by BioLegend (1 mentions)
Alexa fluor 647 anti cd11c, by BioLegend (1 mentions)
Oct medium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti cd206, by Abcam (1 mentions)
Cucl2, by Merck Group (1 mentions)
Radioactive 14c sucrose, by Moravek Biochemicals (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Precellys 24 tissue homogenizer, by Bertin Technologies (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
Cldn5, by Thermo Fisher Scientific (1 mentions)
6 protocols using fluoromount g slide mounting medium
1
Intratumoral Injection and Immunofluorescence
2
Immunofluorescent Analysis of CD204 and CD206
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Sections were deparaffinized and hydrated before antigen retrieval in 10 mM citric acid buffer. Then, sections were incubated in 1% Triton X-100 for 15 min. After eliminating endogenous peroxidase activity with 3% hydrogen peroxide for 15 min, sections were rinsed with PBS and incubated with primary anti-CD204 (cat. no. ab123946; 1 : 50; Abcam Inc.) and anti-CD206 (cat. no. ab125028; 1 : 50; Abcam Inc.) at 4°C overnight. After that, the sections were washed with PBS and incubated with specific secondary antibodies for 30 min at room temperature. Sections were then washed with PBS and sealed with Fluoromount-G™ Slide Mounting Medium (SouthernBiotech, Birmingham, AL, USA) as described before [22 (link)]. Images were taken using a fluorescent microscope and were analyzed using an Image Analysis system, version 11.0.
3
Radioactive Tracer Synthesis and Detection
4
Immunostaining and Fluorescence Imaging of Fly Antennae
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Fly heads were dissected in cold PBT (phosphate buffered saline with Triton X-100) buffer and were fixed in 4% paraformaldehyde (PFA) on nutator at room temperature for 1 hour. Fly heads were washed three times with fresh PBT, and every wash was 10 min at room temperature. Antennae were dissected from heads and were fixed in 4% PFA on nutator at room temperature for 30 min. Antennae were washed three times with fresh PBT, and every wash was 10 min at room temperature. Antennae were mounted using Fluoromount-G Slide Mounting Medium (SouthernBiotech). Images were taken by Olympus FluoView FV1000 confocal microscope. Controls and experimental groups were imaged by same parameters. Native fluorescence was measured by ImageJ. The fluorescence intensity was defined as the fluorescence of region of interest subtracted by that of the background. Statistical tests were performed in GraphPad Prism software. One-way ANOVA was used for significance test, followed by multiple comparisons (compare other groups to group-housed control). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
5
Drosophila Wing Disc Immunostaining
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Larva and forceps were washed in 70% ethanol and PBS. Larva were transferred to dissecting medium (Shields and Sang M3 media, 2% FBS, 1% pen/strep, 3 ng/mL hydroxyecdysone and 0.05 units/l insulin) and their wing discs extracted for up to 15 min. Discs were fixed for 30 min in 4% formaldehyde-PBS at room temperature. Fixed tissue was washed 4x10 min PBT (PBS, 0.3% Triton X-100) and 4x10 min PBT-BSA (PBT, 0.5% BSA). Primary antibody at appropriate concentrations was incubated overnight. Washed were repeated and secondary antibody, with DAPI and Phalloidin incubated for 1-2 h at room temperature. Discs were washed for 3x20 min PBT and 3x quick rinse with PBS. Discs were mounted in Fluoromount G slide mounting medium (Southern Biotech) for imaging.
6
Retinal Immunofluorescence Staining Protocol
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Eyeballs from Ctrl and Sptlc1 ECKO mice were enucleated and fixed in 4% PFA in PBS at room temperature for 20 min. Retinas were isolated under dissection microscopy, permeabilized in 0.5% Triton X-100 in PBS at room temperature for 30 min and blocked with 1% Bovine Serum Albumin (BSA) in PBSt at room temperature for 30 mins. Primary antibodies were added to the sections with indicated dilution in PBSt (NG2 (Sigma, 1:200), Phsopho-histone H3 (BioLegend, 1:400), ESM1 (R and D system, 1:200), LEF1 (Cell Signaling Technology, 1:200), TFRC (Novus Biologicals, 1:200), CLDN5 (Invitrogen, 1:200), MFSD2A (A kind gift from David Silver, 1:200), Alexa Fluor 647 isolectin GS-IB4 conjugate (Invitrogen, 1:500), Alexa Fluor 488-conjugated rabbit monoclonal anti-ERG (Abcam, 1:200), Alexa Fluor 647-conjugated rabbit monoclonal anti-ERG (Abcam, 1:200), Cy3conjugated mouse monoclonal anti-a-smooth muscle actin (Sigma, 1:500)) at 4°C overnight. Retinas were further washed 3 times with PBS for 10 mins at room temperature and incubated with fluorescent conjugated secondary antibodies with 1:500 dilution in PBSt for 2 hours at room temperature. Stained retinas were washed 3 times with PBS for 10 mins at room temperature and mounted using Fluoromount-G slide mounting medium (Southern Biotech). Flat Mount Retina Slides were proceeded to image with confocal microscopy (Zeiss).
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