H0146

10.17504/protocols.io.b5s5q6g6
Cryosectioned, free-floating tissue sections were incubated in blocking buffer (0.5% Triton X-100 (Sigma-Aldrich, T8787–100ML) + 10% normal horse serum (Sigma-Aldrich, H0146–5ML) in 1X PBS) for 1 h at room temperature. Next, sections were incubated in 1 ^o antibody overnight at 4 ^oC then fluorescently conjugated 2 ^o antibody for 1 h at room temperature. Free-floating sections were mounted on SuperFrost + slides then coverslipped with fluorescent mounting medium (Agilent, S302380–2) and #1.5 coverslips.

To confirm respective findings in [⁶⁸Ga]Ga-Pentixafor uptake, immunohistochemistry of CXCR4 was performed on paraffin-embedded sections. Antigen retrieval was performed using proteinase K (E00492, Life Technologies, Vilnius, Lithuania). For peroxidase blocking, slides were incubated in 0.3% H₂O₂. Samples were blocked with blocking horse serum (H0146, Sigma-Aldrich). Tissues were then incubated with a primary monoclonal rabbit (UMB2) anti-CXCR4 antibody (5 µg/mL, 124824, abcam ab, Berlin, Germany) at 4 °C overnight. Then, sections were incubated with a secondary goat anti-rabbit HRP-antibody (1111-035-003, Dianova, Eching, Germany) and with 3.3-diaminobenzindine (DAB) (K346811-2, Dako, Jena, Germany) for visualization. The sample was counter-stained with hematoxylin (Roth T865.1). Subsequently, tissue slides were washed in running tap water. Finally, all slides were mounted with Aquatex (1.08562.0050, Merck, Darmstadt, Germany) for microscope imaging.

Four-micrometer-thick sections from TMA blocks were cut using a microtome, deparaffinized overnight (ON) at 55 °C, and hydrated into milli-Q H₂O through sequential steps of graded ethanol. The immunohistochemistry (IHC) was performed using the EnVision + Dual Link System-HRP (DAB+) kit (Dako #K4065) according to the supplier's instructions. Antigen retrieval was performed with sodium citrate buffer pH 6.0 (Dako #S2031) for 15 min. Endogenous peroxidase activity was quenched followed by unspecific binding blocking with 5% normal horse serum (Sigma Aldrich # H0146) for 1 h at room temperature (RT). For pS727-STAT3 staining, TMAs were incubated ON at 4 °C with primary antibody α-pS727-STAT3 (Cell Signaling #9134) at a final dilution of 1:50. The secondary antibody (labeled polymer-HRP mouse/rabbit) was incubated for 1 h at RT. Finally, HRP detection with DAB-Chromogen and hematoxylin (Sigma Aldrich #HHS32) counterstaining were performed at RT for 20 min and 1 min, respectively. As a negative control, the primary antibody was replaced with non-immune bovine serum (Alpha Diagnostics # 20001-2-1).