Histological and macroscopic evaluation of rectal tissues and biochemical analysis. A) Rectal biopsies (longitudinal cuts) were histologically evaluated by Hematoxilin-Eosin (HE) and Masson’s Tricome stainings in non-CF and CF tissues. Images show healthy epithelia, with no fibrotic processes and only some inflammatory cells were detected. Images shown are representative of the total and correspond to biopsies from a non-CF individual (left) and a CF patient (right) performed with jumbo (3.4 mm Ø) forceps after bowel preparation with glycerol (non-CF) or isotonic saline (CF). In HE-stained sections (top), nuclei are stained in blue and cytoplasm in red. In Tricome’s Masson-stained sections (bottom) collagen (fibrotic biomarker) is stained in blue, nuclei in black, and muscle and cytoplasm in red. Black scale bar represents 250 μM. B) Immunohistofluorescence of rectal biopsies showing nuclei in blue (DAPI staining) and CFTR in green. Images evidence CFTR at the membrane in a non-CF tissue (top panels) and also, albeit weaker, in a biopsy from a CF patient with the F508del/P205S-CFTR genotype (bottom panels). In contrast, a biopsy from a F508del-homozygous CF patient evidences intracellular CFTR staining (middle panels). A negative control (no primary antibody n.c.) was also performed. Scale bar represents 25 μm. C) Western blot of a single rectal biopsy from non-CF individuals (wt-CFTR, lanes 1–2) and from a CF patient with F508del/R334W-CFTR genotype (lane 3) evidence the presence of both immature and mature forms of CFTR (bands B and C, respectively; and from a CF patient with the F508del/F508del-CFTR genotype (lane 4) evidencing only immature form (band B) which is characteristic of the endoplasmic reticulum (ER) and thus corroborating the traffic defect associated with this mutation.