Immediately after excision from mice, some of the tumors were fixed in 5% formalin overnight after which they were dehydrated in ethanol and embedded in paraffin. Tumor sections (5–6μm) were deparafinized, and immunolabeling was performed as described previously (21 (link)). Cells from dissociated HMLEHRASV12 xenografts (both transplantable and non-transplantable) were plated in six well plates and allowed to propagate for 48 hrs. Cells were fixed with 4% paraformaldehyde in 1× PBS for 30 min, washed, and incubated with blocking buffer (TBS pH 7.8, 5% normal goat serum, 1% BSA and 1% triton X-100) for 1hr. The following antibodies were used in our experiments: UCP1 (ab10983, Abcam), UCP2 (AB3226, Millipore, Billerica, MA); and UCP3 (ab3477, Abcam).
Protocol detail
Immunolabeling of Dissociated Xenograft Cells
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Antibodies
Buffer
Cells
Dehydrated ethanol
Embedded paraffin
Formalin
Goat
Mice
Paraformaldehyde
Serum
Triton x 100
Tumor
Ucp1
Xenografts
Corresponding Organization : University of California, Los Angeles
Other organizations : Columbia University, Jackson State University
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Variable analysis
independent variables
- Tumor cells (HMLE^HRASV12^ xenografts)
- Expression of UCP1, UCP2, and UCP3 proteins
- Time of tumor fixation in 5% formalin
- Tumor section thickness (5-6 μm)
- Cell culture duration (48 hrs)
- Cell fixation in 4% paraformaldehyde
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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