Alexa fluor 488 affinipure goat anti mouse

Rabbit polyclonal anti-MCDL and anti-MEIK have been described and characterized previously (Liu et al., 2013 (link); Guo et al., 2017 (link)). Rabbit polyclonal anti-NBCn1 was custom-made against an immunogen “ENAKVTRPNMSPEKPVSVTC” by Genscript (Nanjing, CN). Rabbit anti-NHE3 (cat#ab-95299) and mouse anti-α1 (cat#ab-7671) against the α1 subunit of Na⁺-K⁺ pump (aka Na⁺-K⁺-ATPase) were purchased from Abcam (HK, China). Mouse anti-actin was purchased from Beyotime (Cat#AA128, Haimen, Jiangsu, China). HRP-conjugated secondary antibodies were from Beyotime. Alexa Fluor^® 488 AffiniPure goat anti-mouse (cat# 115-545-003) was from Jackson ImmunoResearch (West Grove, PA, United States). Dylight 549 goat anti-rabbit (cat#E032320-01) was from EarthOx (Millbrae, CA, United States).

In total, 2.5 μm sections of TMAs and paraffinized cell pellets were placed on glass slides and dried for 10 min at 60 °C, dewaxed for 10 min in HistoChoice Clearing Agent (Sigma, H2779), and rehydrated by consecutive washes in ethanol: 100%, 95%, 70%, and 40% v/v for 30 sec each. Heat-induced antigen retrieval was performed at 95 °C for 40 min with Dako Target Retrieval Solution of pH 9 (Dako, S2367). After cooling at room temperature, the slides were incubated 2.5% BSA (Fluka Analytical, 05488) in TBST 0.1% for 1 hour at room temperature. Anti-ADPr [31 (link), 32 ] (rabbit, 1:500 dilution) and anti-ATP5a antibody (Abcam, mouse, 1:250) diluted in the blocking solution were incubated overnight at 4 °C. Secondary antibody Alexa Fluor 488 AffiniPure goat anti-mouse (Jackson ImmunoResearch, 115–545–003, 1:500 dilution) and Cy3 AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, 111–165–144, 1:250 dilution) were incubated for 2 hours at room temperature. Nuclear counterstaining was performed DAPI (Biolegend, 422801, 1:10,000 dilution). Microscope cover glasses (Menzel–Glaser, 7001023) were mounted with Mowiol mounting medium.

Primary antibodies used were: Yap (NB110-58358, NOVUS Biologicals,Littleton, CO, USA), GFAP (MAB360, Merck & Millipore, Billerica, MA, USA), Nestin (MAB353, Merck & Millipore), AQP0 (05-321, Merck & Millipore), Ki67 (ab15580, Abcam, Cambridge, MA, USA), Caspase1 (ab108362, Abcam), β-actin (60008-1-1, Proteintech Group, Wuhan, HB, China), β-Tubulin (CW0098A, CWbiotech, Beijing, China).
Secondary antibodies used were: TRITC AffiniPure Goat Anti-Rabbit (111-025-003, Jackson Immuno-Research, Jennersville, PA, USA), Alexa Fluor 488 AffiniPure Goat Anti-Rabbit (111-545-003, Jackson ImmunoResearch), Alexa Fluor 488 AffiniPure Goat Anti-Mouse (115-545-003, Jackson Immunoresearch), Vectastain Elite ABC kit (PK-4001, ZSGB-BIO).