Vector dab

A cervical cancer tissue microarray (TMA) containing 39 cases of cervical cancer and 9 cases of normal cervical tissue (in duplicate) was purchased from GeneTex, Inc. (GTX21468). Slides were deparaffinised in xylene, rehydrated in a graded series of ethanol solutions and subjected to antigen retrieval in citric acid. Slides were blocked in normal serum and incubated in primary antibody (SUR1 (75-267, Antibodies Inc.)) overnight at 4 °C. Slides were then processed using the VECTASTAIN^® Universal Quick HRP Kit (PK-7800; Vector Laboratories) as per the manufacturer’s instructions. Immunostaining was visualised using 3,3′-diaminobenzidine (Vector^® DAB (SK-4100; Vector Laboratories)). Images were taken using an EVOS® FL Auto Imaging System (ThermoFisher Scientific) at ×20 magnification. SUR1 quantification was automated using ImageJ with the IHC Profiler plug-in [88 (link), 89 (link)]. Histology scores (H-score) were calculated based on the percentage of positively stained tumour cells and the staining intensity grade. The staining intensities were classified into the following four categories: 0, no staining; 1, low positive staining; 2, positive staining; 3, strong positive staining. H-score was calculated by the following formula: (3 × percentage of strong positive tissue) + (2 × percentage of positive tissue) + (percentage of low positive tissue), giving a range of 0–300.

Lung cancer tissue array were purchased from Pantomics, Inc. (Richmond, CA, USA). In brief, slides were treated with high pH antigen retrieval buffer. Rabbit anti-human ABCB4 (1:200; LS-B5729, lifespan biosciences, USA) and normal rabbit serum were used for stainings. Slides were incubated with ABCB4 antibody at 4°C overnight. After extensive washing, sections were incubate with secondary antibody, followed by staining with vector DAB (Vector Laboratories). The slides were washed and counterstained with hematoxylin for 5 min. All slides were analyzed under the Hamamatsu NDP slide scanner (Hamamatsu Nanozoomer 2.0HT) and its viewing platform (NDP. Viewer).

Coronal or sagittal sections (35 µm thickness) were washed with 0.1% Triton in PBS, saturated by incubation with 0.1% Triton in PBS/5% goat serum, and then incubated with primary antibodies as follows: AT8 anti-P-tau pSer202/Thr205 (1/500, Thermo Scientific, #MN1020); anti-P-tau pS396 (1/1000, Abcam, #ab109390); anti-IBA1 (1/1000, Wako, #019-19741), CD68 (1/500, Bio-Rad, #MCA1957) and anti-C1q (1/1000, Abcam, #ab182451). For fluorescent immunostaining, sections were incubated with the appropriate secondary antibody: anti-rabbit IgG Alexa Fluor 488 or 568 (1/2000, Invitrogen); anti-mouse IgG Alexa Fluor 488 or 568 (1/2000, Invitrogen). For non-fluorescent immunostaining, endogenous peroxidase was quenched with PBS containing 3% H₂O₂ for 15 min followed by amplification using the ABC system (VECTASTAIN Elite ABC HRP Kit, Vector Laboratories, Burlingame, CA, USA). Horseradish peroxidase conjugates and 3,3′-diaminobenzidine were used according to the manufacturer’s manual (Vector® DAB, Vector Laboratories, Burlingame, CA, USA). Images were obtained with an Olympus BX61 microscope and analyzed with Fiji software (ImageJ).

Slides (5 µm thick) were autoclaved in Target Retrieval Solution (Dako, S1699, Denmark), incubated in Proteinase K (S3004, Dako, Denmark) or with EDTA- buffer (Prosan, Belgium) according to the immunolabelling for Ki67, CD31, and FITC-dextran, respectively. Endogenous peroxidases were blocked by 3% H2O2/H2O (Merck, Belgium) for 20 minutes, and nonspecific binding was prevented by incubation in PBS/Bovine Serum Albumin 10% (Fraction V, Acros Organics, NJ). Tumor sections were incubated with a mouse monoclonal anti-human Ki-67 antibody (1/100) (clone MIB-1, M7240; DAKO, Denmark), a rat anti-CD31 antibody (1/100) (Ab56299, Abcam, United Kingdom), or a ready-to-use anti-fluorescein antibody (Converter-POD, Roche, France). After 3 washes in PBS or Tris-HCl for CD-31 staining, slides were incubated with an HRP-conjugated secondary antibody, after post antibody blocking (DPVB Blocking, Immunologic NL) for pimonidazole staining, and revealed with Vector DAB (SK-4100, Vector Laboratories, Burlingame, CA, USA). Slides were counterstained with hematoxylin. For lung metastases quantification, six lung sections of 5 µm, spaced by 10 sections of 5 µm, were immunostained with an antibody against human Ki67 as previously described (6 (link)). Metastases were manually counted and classified according to their size (<10 cells, 10 to 50 cells, 50 to 100 cells, >100 cells).