Anti lysozyme c

For light microscopy, tissues and organoids were xed for 16 h in 10% neutral buffered formalin at 4 °C.
The tissues were embedded in para n and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry. Organoids were embedded in Tissue-Tek ® O.C.T. Compound (Sakura Finetek), frozen at -20 °C, and cut into 10-µm-thick sections. For immunohistochemistry, para n sections were prepared by boiling in 10 mM citrate buffer pH 6.0 for 15 min. After blocking with 1% bovine serum albumin, sections were incubated for 16 h at 4 °C with the following primary antibodies: anti-lysozyme C (1:200, goat polyclonal, Santa Cruz Biotechnology); antiproliferating cell nuclear antigen (PCNA, 1:100, mouse monoclonal, Dako); anti-5-mC (1:300, rabbit monoclonal, Cell Signaling Technology); and anti-H3K4, 9, 27, 36, and 79me3 (1:300, rabbit monoclonal, Cell Signaling Technology). Before incubation with secondary antibodies, the tissues were washed to remove the unbound antibodies. The sections were then incubated with secondary antibodies (1:1000 dilution; Alexa Fluor 488 and/or Alexa Fluor 555, Invitrogen) for 1 h at 25 °C and mounted using UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology). The samples were observed under a uorescence microscope (Axioskop 2 plus or Apotome. 2, Carl Zeiss).