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2 protocols using ab55812

1

PrPC-specific antibody characterization

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PrPC-specific antibodies 3F4 and 8H4 were obtained from Signet laboratories (Dedham, MA, USA). 3F4 binds to an epitope on amino acids 109–112 (Human PrP sequence), while 8H4 binds an epitope in the C-terminal amino acids 145–180 of human and mouse PrP62 (link). Other primary antibodies were obtained from the following sources: anti-ferritin (F5012, Sigma Aldrich, USA), anti-DMT-1 (ab55812, Abcam, USA), anti-TfR (ab84036, Abcam, USA), anti-Tf (GTX21223, GeneTex, USA), and anti-β-actin (MAB1501, Millipore, USA) Secondary antibodies conjugated with HRP were from GE Healthcare (anti-mouse, LNA931V, anti-rabbit, LNA934V). Alexa fluor-conjugated secondary antibodies were from molecular probes, ImmPACT DAB (SK4105) from Vector labs, USA, PNGase F (P0704S) from NEB, USA and Lipofectamine 3000 transfection reagent from Invitrogen, USA. Radiolabeled 59FeCl3 was obtained from Perkin-Elmer, USA. siRNA against PrP (sc36318) and scrambled siRNA (sc37007) were purchased from Santa Cruz Biotechnology Inc, USA. For immunohistochemistry of hamster retinal sections PrP-6C2 (CVI-WUR, Lelystad, Netherland), ferritin (Jackson ImmunoResearch, West Grove, PA, USA), and Iba1 (019–19741, Wako Chemicals, USA) antibodies were used. FAC (F5879) and all other chemicals were from Sigma Aldrich, USA.
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2

Immunofluorescence Analysis of Liver Tissue

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Liver tissues were collected and routinely embedded in optimal cutting tissue. Frozen liver samples were sliced into 14-μm sections. For immunofluorescence staining, after being washed with phosphate-buffered saline (PBS) 3 times, the sections were penetrated with methanol at -20ºC for 10 minutes and sealed with 5% goat serum at room temperature for 1 hour. Finally, frozen liver sections were stained with CD68 (ab201340; Abcam, Cambridge, UK), CD11b (ab52478; Abcam), Ptgs2 (ab15191; Abcam), DMT1 (ab55812; Abcam), GPX4 (ab125066; Abcam), F4/80 (DF2789; Affinity, Jiangsu, China), TGFβ1 (ER31210; huabio, Hangzhou, China; 21898-1-AP; Proteintech), Ki67 (9129s; Cell Signaling Technology, Boston, MA), GSK3β (9832s; Cell Signaling Technology), P-GSK3β (5558p; Cell Signaling Technology), Nrf2 (16396-1; Proteintech, Chicago, IL), and iNOS (ab178945; Abcam) overnight at 4°C. After extensive washing, the frozen sections were incubated with the respective fluorescent secondary antibodies. Finally, the nucleus was stained with 4′,6-diamidino-2-phenylindole for 10 minutes.
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