Lcm staining kit

7–9 week old C57BL/6J male mice brains were freshly frozen in OCT compound and sectioned at 14 μM on Leica 3050 s cryostat, taking every other section throughout the hippocampus. Sections were then mounted on PEN membrane slides, ~12 sections per slide with 3 slides per brain. The staining and preparation of sections for the LCM procedure was done using the LCM staining kit (Cat# AM1935, Life Technologies) with Cresyl Violet following manufacturer's recommendations. Following staining, slides were kept at room temperature before beginning LCM. The microdissection of the CA1, CA3 and DG hippocampal regions were done using the Arcturus PixCell II LCM microscope where the laser power was set at 50 mW and each capture was done with 2 or fewer laser pulses. After dissection, each sample was placed in 50 μl ice-cold Trizol and RNA was extracted using the Trizol-Chloroform method (Life technologies). Purified RNA from each dissection was subjected to a single round of linear T7 RNA Polymerase–driven transcription using MessageAmp™ II aRNA Amplification Kit (Cat# AM1751, Life Technologies).

The laser capture microdissection was carried out as described in Kadakkuzha et al. [47 (link)]. Nine-week-old C57BL6/J mice underwent contextual fear conditioning. One hour later, the mice were sacrificed, and the brains were freshly frozen in OCT compound. The brains were then sectioned at 14 µm on a Leica 3050 s cryostat, saving every other section throughout the prefrontal cortex. Sections were immediately mounted unto PEN membrane slides. Next, the sections were stained with Cresyl Violet using the LCM staining kit (Cat# AM1935, Life Technologies) according to the manufacturer’s instructions. Following staining, the slides were stored at room temperature until the laser capture microdissection (LCM) procedure. Microdissection of the PrL and IL were performed using the Leica LMD7000 LCM microscope, in which laser power was set at 50 mW and each capture was performed with two or fewer laser pulses. After the microdissection, each sample was placed in 50 µl of ice-cold lysis buffer. The RNA was extracted using the Arcturus™ PicoPure™ RNA Isolation Kit (Cat#1220-01, Applied Biosystems) according to the manufacturer’s instructions.

One hour after finishing the training in CFC or MWM, mice were sacrificed by a fast decapitation and the brains were removed, briefly washed in D-PBS, placed in cryomolds with OCT and frozen in dry ice and stored at −80°C (Kadakkuzha et al., 2015). Fresh frozen brains were coronal sectioned at 14μm on a Leica 3050s cryostat at −20°C after 1h of acclimation to this temperature. Dorsal hippocampal sections were mounted on PEN membrane slides (Leica), ~12 sections per slide with 3 slides per brain from a total of 6 brains. Staining and preparation of sections for the Laser Capture Microdissection (LCM) procedure was done using LCM staining kit (Life Technologies) with Cresyl Violet following manufacturer’s recommendations. Following staining and dehydration, slides were kept at room temperature for 15 min before LCM. The microdissection of the CA1 and CA3 hippocampal regions were done using the Leica LCM microscope (Leica LMD7000) where the laser power was set at 60 mW. Tissue was collected in RNAse free microtubes were 50μl of pre-chilled trizol were added just after finishing the micro sectioning. RNA was extracted using the trizol-Chloroform method and stored at −80°C before use.