Immunostaining of Intestinal Tissue Sections

Neonatal and post-neonatal (0–3 weeks old) intestine was fixed in 10% formalin and embedded in paraffin, whereas post-weanling and adult intestine were fixed and embedded as described previously^{27 (link)}. Briefly, tissue was fixed in 1% paraformaldehyde, washed with 50 mM NH₄Cl, cryoprotected in 30% sucrose (wt/vol) and embedded in Optimal Cutting Temperature (OCT, Tissue-Tek) medium. Immunostaining of frozen or FFPE sections (5–7 μm) was performed using the rabbit anti-laminin (Sigma-Aldrich) or biotin-labeled anti-GFP (Abcam), followed by Alexa Fluor 594 goat anti-rabbit IgG (H+L), Alexa Fluor 647 phalloidin, Alexa Fluor 647 Streptavidin (Invitrogen) and/or Hoechst 33342 dye (Invitrogen). Slides were mounted with ProLong Glass (Invitrogen) and images were acquired on an inverted DMi8 microscope (Leica) equipped with a CSU-W1 spinning disk, ZYLA SL150 sCMOS camera (Andor), PL APO 40x/0.85 dry objectives, and iQ3 acquisition software (Andor). The number of GFP⁺ cells per 0.1 mm² villus was quantified by an observer blinded to the condition.