Elite vector stain abc system

Gramicidin, nigericin, tributyltin, valinomcycin, TMZ, and BMT were purchased from Sigma Chemicals (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), accutase, goat anti-IgG secondary antibodies Alexa Fluor^® 488 and 546, PBFI-AM, calcein-AM, MQAE, and pluronic acid were obtained from Invitrogen (Carlsbad, CA). Elite Vector Stain ABC System and 3-3′-diaminobenzidine (DAB) were from Vector Laboratories (Burlingame, CA). Rabbit anti-WNK1 (t-WNK1) (N-20, #sc-20468) was from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-phospho-WNK1 (p-WNK1) (T60, #AF4720) was from R&D Systems (Minneapolis, MN). Sheep anti-WNK3 (S156C) was developed previously described [35 (link)]. Mouse anti-NKCC (T4) antibody was from Developmental Studies Hybridoma Bank (Iowa City, IA). Mouse anti-α-tubulin (#2125), rabbit anti-ezrin (#3145), rabbit anti-ezrin/radixin/moesin (ERM) (#3142) and rabbit anti-phosphorylated-ERM (#3149) antibodies were from Cell Signaling (Beverly, MA). Rabbit anti-phosphorylated-NKCC1 (p-NKCC1) antibody (R5) was a kind gift from Dr. Biff Forbush (Yale University). Sheep anti-phosphorylated-NKCC1 recognizing the same residues (Thr²¹² and Thr ²¹⁷) as R5 was developed as previously described [35 (link)]. Rabbit anti-SPAK/OSR1 (t-SPAK/t-OSR1) and rabbit anti-phosphorylated-SPAK (Ser³⁷³)/OSR1 (Ser³²⁵) (p-SPAK/p-OSR1) were developed as described before [36 (link),37 (link)].

Paraffin-embedded, 1-mm formalin fixed tissue sections were mounted on microscope slides and processed as previously described. Immunohistochemical staining of anti-human CEBPD (1:200, Biorbyt, orb213725) was performed on tissue sections. The sections were treated with a heat-induced epitope retrieval technique using a citrate buffer at pH 6.0. The sections were then blocked for endogenous peroxidase and biotin before incubation with primary antibodies for 3 h at room temperature. The Elite Vector Stain ABC System (Vector Laboratories) was used as the detection system and diaminobenzidine as the chromogen. Nuclei were counterstained with hematoxylin. The immunoreactivity score of CEBPD was determined by multiplication of the values of the percentage of CEBPD positive cells (0: 1%, 1: 1–25%, 2: 26 –50%, 3: 51–75%, 4: 75%) and the values for CEBPD staining intensity (0: no staining, 1: weak staining, 2: moderate staining, 3: strong staining), as described previously [27 (link)].

IHC was conducted on paraffin embedded, formalin fixed tissue sections (FFPE) of human lung to brain metastatic cancer biopsies using antibodies to CD15s and CD62E. Briefly, 4 μm thick FFPE sections were dewaxed in Xylene, rehydrated in 100%, 95%, 70% ethanol and deionized water respectively followed by 40 min of heat-induced epitope retrieval with citrate buffer (6.0 pH) at 95 °C and incubated with 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase and biotin activity. Tissues were then blocked with 3% horse serum for 30 min and probed with primary antibodies for 1 h at room temperature. The Elite Vector Stain ABC system (Vector Labs, Burlingame, CA, USA) was employed as a detection system and DAB stain (Vector Labs, Burlingame, CA, USA) was used as a chromogen. Meyer’s haematoxylin (Sigma-Aldrich, Gillingham, UK) was used as a counterstain. Mouse IgM and IgG isotopes were used instead of primary antibodies in negative controls. Tissue sections were examined using a bright field automated microscope Ariol (Leica) for qualitative image analysis.