Dab chromogen solution

Sagittal sections through E15 and adult rat brain including lateral choroid plexus were selected from the collection of rat tissue at the Faculty of Health and Medical Sciences, University of Copenhagen and used for immunohistochemical detection of SLC16a10, Slc38a5 (SNAT5), Slc4a1, Slc11a1 (NRAMP), Slc39a4 (ZIP4), and Slc1a3 (EAAT1). Sections were deparaffinised in xylene, rehydrated through graded alcohols followed by treatments in 0.5% hydrogen peroxide in methanol for 15 min and rinsing in TRIS buffered saline (TBS) as described previously (Liddelow et al., 2012 (link)). Following removal of non-specific binding by incubation for 30 min with blocking buffer (ChemMate antibody diluent S2022, DakoCytomation, Glostrup, Denmark) at room temperature sections were incubated in primary antibodies as listed in Table 1.
After overnight incubation sections were washed in TBS and incubated for 30 min in EnVisionTM+ System/HRP (DAKO), K5007, for rabbit antibodies and RPN1025 from GE Healthcare for donkey anti-goat antibodies and then Vector: Vectastain RTU elite ABC reagent PK 7100. This was followed by 6 min incubation with DAB-chromogen solution (DAKO) and counterstaining with Mayer's haematoxylin, dehydrated and mounted with DPX. Control sections contained no primary antibodies and were always blank.

For cross-section studies, histological macrosections of 5 μm in thickness were cut from the FFPE blocks of each prostate (A,B and C), next to the HE-stained sections. Paraffin was removed with xylene and the sections were rehydrated with series of alcohol. Antigen retrieval was carried out by microwaving the slides in Target Retrieval solution (Dako) at pH 9 for 7 min. The tissue sections were incubated for one hour with rabbit monoclonal AMACR (P504S) antibody (1:200, clone 13H4, Zeta Corporation). The primary antibody was detected with EnVision + Dual Link System-HRP (Dako) and visualized with DAB+ chromogen solution (Dako). The slides were observed by an experienced uropathologist using a Leica DM3000 light microscope equipped with Leica DFC 420 digital camera and Leica Application Suite version 2.5.0 R1 (Leica Microsystems, Wetzlar).

The cultures of PDLSCs and PSCs were incubated with antibodies to CD73 (MA5-15537, Invitrogen, Waltham, MA, USA); CD90 (ab133350, Abcam, Cambridge, UK); STRO-1 (39-8401, Invitrogen). Deparaffinized sections were incubated with antibodies to osteopontin (OPN, ab218237. Abcam), osteocalcin (OC, ab198228, Abcam), and dentin sialophosphoprotein (DSPP, ab216892, Abcam). Prior to immunohistochemical staining, antigen retrieval was performed using Dako PT Link (Dako, Denmark A/S) at 97 °C for 20 min. The unmasking was performed under low pH using EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S). Endogenous peroxidase and host IgG were blocked, and the primary antibodies (dilution 1:200) incubated for 12 h at 4 °C. The EnVision FLEX Detection System (Dako, Denmark A/S) with the chromogen 2, 3-diamino-benzidine DAB (DAB Chromogen Solution, Dako) was used for imaging. Hematoxylin was used to counterstain nuclei. Incubation without primary antibodies was used as a negative control.

The formalin-fixed and paraffin-embedded tissues were cut into sections at a thickness of 4 μm. Antibodies were incubated overnight at 4°C at the following dilutions: 1:200 for CCDC106 (Abcam, Cambridge, MA, USA); 1:100 for p53 (Cell Signaling Technology, Danvers, MA, USA); 1:200 for p21 (Proteintech, Wuhan, China) and 1:500 for ATF4 (Proteintech, Wuhan, China). A biotin-labeled secondary antibody (Ultrasensitive; MaiXin, Fuzhou, China) was incubated at room temperature for 30 min, and color was developed using 3,3’-diaminobenzidine (DAB) Chromogen solution (Dako, Tokyo, Japan).
The expression of CCDC106, p53, p21 and ATF4 was scored according to the number of positively stained cells and their staining intensity. Expression was divided into four grades according to the color intensity: 0 (no color), 1 (light yellow particles), 2 (medium strength yellow particles) and 3 (dark yellow or tawny particles). According to the staining area of cells, there were four grades: 1 (1%-25%), 2 (26%-50%), 3 (51%-75%) and 4 (76%-100%). A final score of 0–12 was obtained by multiplying the intensity and percentage scores.