Table 1.

A list of the specimens tested for FISSEQ library construction.

Types	Fixation	Mounting substrate	Permeabilization	Notes
HeLa, 293A, COS1, U2OS, iPSC, primary fibroblasts, bipolar neurons	10% formalin or 4% PFA	Poly lysine- coated cover slip (Matrigel for iPSCs)	70% EtOH or 0.25% Triton- X100 (0.1N HCl optional)	Changes in temperature can cause altered mRNA localization
Mouse embryo FFPE section (20-um)	Already fixed	Superfrost Plus glass slide	0.1% Pepsin in 0.1N HCl	Use silicone isolators (Grace Bio-Labs)
Mouse brain fresh frozen section (20-um)	10% formalin	Poly lysine- coated cover slip	0.1% Pepsin in 0.1N HCl	Use silicone isolators (Grace Bio-Labs)
iPS-derived 3D organoids	10% formalin	Poly lysine- coated cover slip (embed in Matrigel & fix with 4% PFA)	0.25% Triton- X100 & 0.1N HCl	10% formalin less effective for fixing Matrigel
Dechorionated whole mount Drosophila embryos	10% formalin	Poly lysine- coated cover slip (embed in Matrigel & fix with 4% PFA)	100% MeOH, then PBS with 0.2% Triton- X100 & 0.2% Tween-20	10% formalin less effective for fixing Matrigel