Extended data Figure 2-1
Postimaging immunohistochemical analysis of histology. After imaging, sagittal slices (n = 22 slices, 100 μm, from 6 mice) were fixed with 4% PFA after the bioluminescence recording, and immunohistochemically labeled with a cocktail of antibodies against AVP (AVP-NP, PS419) and VIP (Peptide Institute, 14110). Immunohistochemical staining was examined by fluorescent microscopy (BZ9000; Keyence) as previously reported (Yoshikawa et al., 2015). A, Schematic drawing of the SCN. The blue lines indicate the plane of 100-μm sagittal slices that were made. Numbers (–2 ∼ +2) indicate position of the sagittal slices with respect to the midline. B, Images of bioluminescence, immunohistochemical staining for AVP, VIP, and overlay of AVP and VIP. C, Chart comparing robustness of oscillation and expression of AVP and VIP in each slice. Slice IDs indicate good (black) and poor (grey) rhythms. Number of immunopositive cell bodies for VIP and AVP are expressed in symbols. ++, high; +, medium; ±, low; –, none. Robust oscillation requires both AVP and VIP expression. Slices bearing a large number of AVP neurons but lacking VIP showed poor rhythm. The one slice that had both peptides could not classified with respect to rhythmicity due to technical equipment problems. Anatomical analysis of peptide expression was conducted independently and prior to classification of rhythmicity. The number of positive cell bodies were scored as follows. For VIP: >10 cells = ++, 5–9 cells = +, 1–4 cells = ±, no cells = –. For AVP: >20 cells = ++, 5–20 cells = +, 1–5 cells = ±, no cell = –. Download Figure 2-1, TIF file.