Tissue tek oct

Male and female mice were randomly divided into two cohorts. One cohort was terminally sacrificed at 6 months of age (young), while the second were designated for aging. Animals designated for aging underwent survival biopsies at 14 months of age (middle age) as previously reported [20 (link)] under aseptic conditions as approved by the University of Washington Institutional Animal Care and Use Committee. For terminal kidney necropsies at 6 months of age (n=9) or 20-24 months of age (aged, n=14), mice were killed with an overdose of Ketamine (JHP Pharmaceuticals LLC, Rochester, MI)/Xylazine (Patterson Veterinary, Devens, MA)as previously described [27 (link)]. Kidney tissue was butterflied and one half butterfly was placed into 4% paraformaldehyde (PFA, Affymetrix, Santa Clara, CA) in PBS for 45 minutes, washed briefly in PBS, placed in 30% sucrose (Sigma-Aldrich, St Louis, MO) in PBS overnight, blotted dry, embedded in Tissue-Tek® O.C.T. Compound (VWR, Radnor, PA), and frozen in a 100% ethanol/dry ice bath. The second half of the butterflies were placed into 10% neutral buffered formalin (NBF) (Globe Scientific, Mahwah, NJ) overnight at 4°C, transferred to 70% ethanol, and embedded in paraffin.

For immunohistochemistry, cleaned reproductive tracts (see above) were fixed, in toto, in 4% methanol-free paraformaldehyde (Polysciences, Warrington, PA) in mHTF-Hepes overnight at 37 °C. After fixation, reproductive tracts were washed 3x in sterile PBS, air dried at -20 °C for 2–4 hrs, and embedded in Optimal Cutting Temperature compound (Tissue-Tek OCT; VWR, Bridgeport, NJ). Embedded reproductive tracts were stored at -80 °C. For processing, 7-μm sections were cut on a cryostat at −20 °C (CM1850 UV cryostat; Leica, Buffalo Grove, IL), collected on clean glass slides (Diamond White Glass 25 × 75 × 1 mm, (+)- charged; MidSci, Valley Park, MO), and stored at −80 °C until staining. To detect direct GFP expression, slides were brought to room temperature for 5 minutes and stained for 10 min with 10-μg/ml Hoechst 33342. Sections were sealed in antifade solution using a clean coverslip before imaging. In some instances, GFP CETN2 sections were first immunostained using antibodies to Ak15 γ-tubulin (1:500; Sigma-Aldrich) and microtubules (YOL1/34; 1:200; EMD Millipore) as describe above.

For PCR analysis of rearranged immunoglobulin genes, macroscopically visible tumors were dissected and cryofixated in Tissue Tek® O.C.T.™ (VWR, Cat# SAKU4583) on dry ice.
DNA was isolated from 10 to 20 8‐µm frozen tissue sections of biopsies from humanized mice using the PureGene DNA Isolation Kit (Qiagen, Hilden, Germany). PCR was performed with six framework region 1 subgroup‐specific primers and a JH primer mix (3′ JH mix) for 35 cycles in six separate reactions, using 300 ng of DNA per reaction. Primer sequences have been published before (Kuppers et al, 2013 (link)). PCR products were purified from agarose gels and Sanger sequenced with the IGHV primers used for PCR and the BigDye Sequencing Kit (ABI, Heidelberg, Germany) on an Applied Biosystems 3130 Genetic Analyzer (ABI). Sequences were evaluated with the IMGT/V‐Quest software (http://www.imgt.org/IMGT_vquest/input).