The 3D spheroid cocultures were kept in 4% paraformaldehyde at 4 °C for 30 min. The specimens were then processed, embedded in paraffin and sectioned at 5 μm, essentially as we did before [60 (link), 61 (link)]. Heat-induced antigen retrieval was performed in citrate buffer using a pressure cooker. Sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies used in this study were P2RY12 (BioLegend, CA) and Nestin (Rat401, DSHB) as we used in previous studies. Sections were then incubated with FITC- or TRICT-conjugated IgG (for 1 h at room temperature), followed by DAPI staining (Vector Laboratories). Images were acquired with Olympus BX41 microscope. Six random fields from three different tissues in each group were quantified using the ImageJ software.
Protocol detail
Immunohistochemical Analysis of 3D Spheroid Cocultures
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Antibodies
Antigen
Buffer
Citrate
Cocultures
Dapi
Embedded paraffin
Fitc
Microscope
Nestin
Paraformaldehyde
Pressure
Tissues
Vector
Corresponding Organization :
Other organizations : University of Cincinnati Medical Center
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Variable analysis
independent variables
- None explicitly mentioned
- Expression of P2RY12 and Nestin proteins in 3D spheroid cocultures
- Paraformaldehyde concentration (4%)
- Paraformaldehyde incubation time (30 min)
- Tissue sectioning thickness (5 μm)
- Antigen retrieval method (heat-induced in citrate buffer using a pressure cooker)
- Primary antibody incubation (overnight at 4 °C)
- Secondary antibody incubation (1 h at room temperature)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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