Pen membrane glass slides

Thirty‐month‐old male Fischer 344 x Brown Norway F1 hybrid rats (Rattus norvegicus) were purchased from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN, USA). β‐guanidinopropionic acid was synthesized as described (Herbst et al., 2013)] from cyanamide and β‐alanine. GPA was purified by recrystallization and the synthesis confirmed by mass spectrometry and IR spectroscopy. β‐GPA was formulated to 1% by weight in 6% fat rodent chow (Harlan‐Teklad, Madison, WI, USA) and fed for 4 months ad libitum. Rats were housed on a 12‐h light/dark cycle. No significant difference was observed in the survival of rats treated with β‐GPA vs controls. Animals were euthanized, the quadriceps muscles dissected from the animals, weighed, bisected at the mid belly, embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), flash frozen in liquid nitrogen, and stored at −80 °C. A minimum of one hundred 10‐μm‐thick consecutive transverse cross sections were cut with a cryostat at −20 °C and placed on Probe‐On‐Plus slides or PEN membrane glass slides (Life Technologies, Carlsbad, CA, USA) for laser microdissection. Slides were stored at −80 °C until needed.

The TAMU, College of Veterinary Medicine & Biomedical Science histology core performed transverse cryo-sectioning (10 μm) of tick attachment biopsies using Leica CM1850 UV Cryostat (Deer Park, IL). The rationale for transverse sectioning is that the location of tick mouthparts in the section guided the location of the tick attachment cement. Sectioning of biopsies began from the tick side and proceeded along the length of the mouthpart and finishing at the tick-feeding cavity where tick mouthparts were absent. Cryosections were placed onto PEN membrane glass slides (Life Technologies, Austin, TX, USA) which allowed for efficient recovery of dissected sections onto sample caps. Slides were stored in a slide box with a desiccant at – 80 °C until laser capture microdissection.