Rat anti cd34

For routine immunostaining, back skins were embedded and frozen on dry ice in OCT compound (Sakura Finetek). For detection of GFP in tissue sections, back skins were pre-fixed with 4% PFA at room temperature for 2 hrs, washed with PBS, soaked in 10 % sucrose at room temperature for 2 hrs and then in 30 % sucrose at 4°C overnight; skins were then equilibrated with 30 % sucrose/OCT (1:1) at room temperature for 6 hrs and embedded and frozen on dry ice in OCT compound.
Skin sections (6–9 μm) were fixed in 4% PFA and stained with the following antibodies: rabbit anti-neurofilament (AB5539; Fisher Scientific) 1:500, rabbit anit-S100B (Z0311; Daco) 1:500, rabbit anti-Lgr6 (IMGENEX; IMG-71579) 1:100, rabbit anti-GFP (Molecular Probes; G10362) 1:300, rat anti-cd104 (β4 integrin) (BD Pharmingen; 553745) 1:200; rat anti-cd34 (BD Pharmingen; 553731) 1:300.

The frozen-cut skin sections were incubated with primary antibodies in the blocking solution for 1-4 hrs at room temperature or overnight at 4°C with following dilution: rabbit anti-Runx1 (1:4000, a gift from Thomas Jessell), mouse anti-Myc tag (1:200, Zymed), rat anti-CD34 (1:50, BD Biosciences), rabbit anti-K5 or K14 (1:1000, Covance), rabbit anti-activated Caspase-3 (1:500, R&D Systems), mouse anti-AE13 (1:50, Immunoquest), mouse anti-AE15 (1:10, a gift from Tung-Tien Sun), mouse anti-GATA3 (1:100, Santa Cruz Biotechnologies), rat anti-BrdU (1:300, Abcam), rabbit anti-Ki67 (1:1000, NovoCastra Laboratories). This was followed by incubation in secondary antibody fluorescently coupled with FITC or TxRed.

Immunohistochemistry on lung sections was performed as previously reported (Wenes et al., 2016 (link)). In brief, for serial sections of lungs from FASN cut at 7 μm thickness, tissue samples were fixed in 2% PFA overnight at 4°C, dehydrated and embedded in paraffin. Paraffin slides were first rehydrated to further proceed with antigen retrieval in citrate solution (DAKO). Sections were then fixed in 100% methanol. If necessary, 0.3% hydrogen peroxide was added to methanol, to block endogenous peroxidases. The sections were blocked with the appropriate serum (DAKO) and incubated overnight with the following antibodies: rabbit anti-FASN (Abcam ab 99359), rat anti-CD34 (BD Pharmingen), rabbit anti-phoshpo-p70S6K (T389) (Cell Signaling, 9205), rabbit anti-phospho-4EBP1 (Cell Signaling 2855. Appropriate biotin-labeled secondary antibodies (Jackson Immunoresearch) 1:300 were used, along with streptavidin-bound peroxidase. When necessary, TCA fluoresceine-tyramine or TSA Plus Cyanine 3 system amplification (Perkin Elmer, Life Sciences) were performed according to the manufacturer’s instructions. The sections were subsequently stained with Hoechst. ProLong Gold mounting medium without DAPI (Invitrogen) was used. Microscopic analysis was done with an Olympus BX41 microscope and CellSense imaging software.