Tumor inoculation and treatment were performed following the timeline in Fig. 1a . For hematoxylin and eosin staining, mice were sacrificed 3 days after the second treatment, and tumors were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. For immunofluorescence, tumors were isolated on day 14 and processed as previously detailed64 (link). Flank tumors were used to analyze infiltrate by immunofluorescence in the BRafCA PtenloxP Tyr::CreERT2 model to allow for higher quality sectioning. For staining, anti-CD8 (CT-CD8a, Cedar Lane), DAPI, and phalloidin were used. The images were acquired using an Olympus Fluoview FV1200 microscope equipped with 10X (NA 0.40) and 30X (NA 1.05) objectives and optimum lasers and filter sets. The images were acquired under identical acquisition setting and subsequently processed using Fiji image analysis software.
Protocol detail
Immunofluorescence analysis of tumor infiltrates
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Dapi
Embedded paraffin
Eosin
Formalin
Immunofluorescence
Mice
Microscope
Phalloidin
Timeline
Tumor inoculation
Tumors
Corresponding Organization :
Other organizations : Massachusetts Institute of Technology, Stanford University
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Variable analysis
independent variables
- Tumor inoculation
- Treatment
- Tumor infiltrate analyzed by immunofluorescence
- Mice were sacrificed 3 days after the second treatment
- Tumors were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin
- Tumors were isolated on day 14 and processed as previously detailed
- Flank tumors were used to allow for higher quality sectioning
- Anti-CD8 (CT-CD8a, Cedar Lane), DAPI, and phalloidin were used for staining
- Images were acquired using an Olympus Fluoview FV1200 microscope with 10X (NA 0.40) and 30X (NA 1.05) objectives and optimum lasers and filter sets
- Images were acquired under identical acquisition settings and subsequently processed using Fiji image analysis software
- No positive or negative controls were explicitly mentioned in the input protocol.
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