Frozen Skin Tissue Block Preparation: A Protocol to Preserve Cutaneous Melanoma Samples from Murine Dorsal Skin

Abstract

Source: Moon, H., et al. Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells. J. Vis. Exp. (2019)

This video describes the detailed protocol for isolating melanoma-containing murine dorsal skin, followed by cryo-embedding to prepare a frozen skin tissue block. The cryo-embedded tissue block can be sectioned and microscopically examined to study melanoma progression.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Tissue Processing Skin isolation Obtain the following prior to starting: necropsy instruments, filter paper, and paper towels. Euthanize the mice according to approved protocols, and confirm death prior to proceeding. Using a hair clipper, shave any hair from the dorsal skin …

Representative Results

Figure 1: Skin tissue isolation and cryo-embedding. Following euthanasia, shave the mouse dorsal skin region of interest and embed skin in cryomold containing OCT. The dotted line on the mouse represents the midline. Typically, 3 or 4 pieces of skin can be isolated with line segment 3-4 along the midline; one such piece is indicated by a rectangle 1/2/3/4. These skin pieces should be embedded in …

Materials

Pet hair trimmer	Wahl	09990-502
Hair removal cream	Nair	n/a	Available at most drug stores
Cotton swabs	various
Ultraviolet light bulb	UVP	95-0042-08	model XX-15M midrange UV lamp
200 proof ethanol	various		pure ethanol
Histoplast PE	Fisher Scientific	22900700	paraffin pellets
Neutral Buffered Formalin, 10%	Sigma	HT501128-4L
O.C.T. Compound	Thermo	23730571
Tissue Cassette	Sakura	89199-430	for FFPE processing
Cryomolds	Sakura	4557	25 x 20 mm
FFPE metal mold	Leica	3803082	24 mm x 24 mm
Isoflurane	various		Veterinary grade
Anesthesia inhalation system	various		Veterinary grade
Fine scissor	FST	14085-09	Straight, sharp/sharp
Fine scissor	FST	14558-09	Straight, sharp/sharp
Forcep	FST	11252-00	Dumont #5
Forcep	FST	11018-12	Micro-Adson
Tyr-CreER; LSL-BrafV600E; Pten f/f	Jackson Labs	13590
LSL-tdTomato	Jackson Labs	7914	ai14