Figure 2. TCA and PFA fixation create differences in NC-specific transcription factor fluorescence levels across nuclei.

IHC of definitive NC cell markers SOX9, PAX7, and SNAI2 in stage HH9–10 chicken embryos fixed in (A, D, G, J) 4% PFA for 20m, (B, E, H, K,) 2% TCA for 1h, and (C, F, I, L) 2% TCA for 3h. (A-C) Wholemount chicken embryos after IHC for PAX7 (purple) and DAPI staining (cranial region, anterior to the right). Transverse sections from embryos after IHC for (D-F) SOX9 (G-I), PAX7, and (J-L) SNAI2. (M-P) High magnification images of 2% TCA 1 hr fixed embryo nuclei after IHC for PAX7 and SNAI2 with DAPI staining. (Q) Graph showing relative fluorescence intensity of DAPI across the nucleus comparing the three fixative conditions shows relatively uniform fluorescence across the nuclei, n=5. (R) Graph showing relative intensity of SOX9 across nuclei comparing the three fixative conditions shows some variation in signal across the nuclei with variable peaks in intensity in PFA fixation, n=5. (S) Graph showing relative fluorescence of PAX7 across the nucleus comparing the three fixative conditions shows similar trends in intensity across nuclei in all fixatives, n=5. (T) Graph showing relative fluorescence of SNAI2 across the nucleus comparing the three fixative conditions show distinct fluorescence peaks in TCA fixatives, n=5. Fixation with 2% TCA for 1h increased visualization of nuclear spatial arrangement. The scale bar for the wholemount is 100 μm, the transverse section is 50 μm, and the high magnification transverse section is 20 μm.