Journal: Open biology
Article Title: Sex-specific loss of mitochondrial membrane integrity in the auditory brainstem of a mouse model of Fragile X Syndrome.
doi: 10.1098/rsob.240384
Figure Lengend Snippet: Figure 3. (A) Representative fluorescent micrographs and Fiji-based image analysis pipeline to automatically extract mitochondrial integrity from confocal images using the JACoP plugin [43]. Once TOMM20- (pseudocolour cyan) and PMPCB- (pseudocolour magenta) positive xy individual optical sections were obtained as in figure 1, Mander’s M1 and M2 coefficients were calculated using the JACoP plugin. The performance of these calculations was controlled with Costes’ automatic threshold, randomization and with a cytofluorogram directly with JACoP built-in solutions, and this for every pair of images analysed. Scale bar: 10 µm. (B) Violin plots of Mander’s M1 and (C) of Mander’s M2 coefficients in male and female mice, either wild-type (WT) or KO for Fmr1. All analyses were performed as in (A) n = 90 MNTB neurons from three WT males, 60 MNTB neurons from three WT females, 120 MNTB neurons from five Fmr1 KO males and 90 MNTB neurons from three Fmr1 KO female mice. Data extend from min to max. Individual n measurements are shown as dots in each condition and animals represented by lines of dots (dark purple Fmr1 KO females, darker green B6 wild-type females, lighter purple Fmr1 KO males and lighter green B6 wild-type males). Values span from 0 to 1, where 0 indicates no colocalization and 1 indicates perfect colocalization. *p ≤ 0.05; all other comparisons were not significant. (D) Representative fluorescent micrographs of MNTB neurons co-stained with antibodies against the OMM marker TOMM20 (pseudocolour cyan) and the IMM/matrix marker PMPCB (pseudocolour magenta), in wild-type (WT) or Fmr1 KO female mice. TOMM20 and PMPCB were merged to show partial marker overlap. Dotted area: magnified region of interest (ROI) to illustrate membrane integrity loss in Fmr1 KO mice compared with wild-type. Arrows show TOMM20-positive, but PMPCB-negative mitochondria. The dotted arrow shows PMPCB-positive, but TOMM20-negative mitochondria. Scale bar: 10 µm. A.U.: arbitrary units.
Article Snippet: Following blocking, sections were incubated with an anti-PMPCB polyclonal antibody raised in rabbit (Proteintech cat. no. 16064-1-AP, RRID:AB_2167122) and used at a 1 :1 000 dilution, with an anti-TOMM20 (Abcam cat. no. ab56783, RRID: AB_945896) monoclonal antibody raised in mouse and used at a 1 : 5000 dilution, and 1% NGS in AB media without Triton-X overnight at 4°C on a rotating shaker.
Techniques: Staining, Marker, Membrane
Proteintech is a verified supplier 
![Figure 2. (A) Fiji-based image analysis pipeline to automatically extract mitochondrial length, branching and the overall number of mitochondria from confocal images. Once PMPCB-positive, xy individual optical sections were obtained as in figure 1 (representative micrograph, pseudocolour magenta) (1), the signal is skeletonized (2) to obtain mitochondrial objects (3). Scale bar: 10 µm. For every object, the aspect ratio and the form factor were calculated following [42,44]. The overall number of PMPCB-positive objects of ≥15 px was also extracted. (B) Violin plots of aspect ratio and (C) form factor analyses in male and female mice, either wild-type (WT) or KO for Fmr1. All analyses were performed as in (A) n = 90 MNTB neurons from three WT males, 60 MNTB neurons from three WT females, 120 MNTB neurons from five Fmr1 KO males and 90 MNTB neurons from three Fmr1 KO female mice. Data extend from min to max. Each vertical line of dots represents data from individual animals with individual n measurements shown as dots in each condition (dark purple Fmr1 KO females, darker green B6 wild-type females, lighter purple Fmr1 KO males and lighter green B6 wild-type males). A.U.: arbitrary units.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_9994/pm40359994/pm40359994__page5_image1.jpg)
![Figure 3. (A) Representative fluorescent micrographs and Fiji-based image analysis pipeline to automatically extract mitochondrial integrity from confocal images using the JACoP plugin [43]. Once TOMM20- (pseudocolour cyan) and PMPCB- (pseudocolour magenta) positive xy individual optical sections were obtained as in figure 1, Mander’s M1 and M2 coefficients were calculated using the JACoP plugin. The performance of these calculations was controlled with Costes’ automatic threshold, randomization and with a cytofluorogram directly with JACoP built-in solutions, and this for every pair of images analysed. Scale bar: 10 µm. (B) Violin plots of Mander’s M1 and (C) of Mander’s M2 coefficients in male and female mice, either wild-type (WT) or KO for Fmr1. All analyses were performed as in (A) n = 90 MNTB neurons from three WT males, 60 MNTB neurons from three WT females, 120 MNTB neurons from five Fmr1 KO males and 90 MNTB neurons from three Fmr1 KO female mice. Data extend from min to max. Individual n measurements are shown as dots in each condition and animals represented by lines of dots (dark purple Fmr1 KO females, darker green B6 wild-type females, lighter purple Fmr1 KO males and lighter green B6 wild-type males). Values span from 0 to 1, where 0 indicates no colocalization and 1 indicates perfect colocalization. *p ≤ 0.05; all other comparisons were not significant. (D) Representative fluorescent micrographs of MNTB neurons co-stained with antibodies against the OMM marker TOMM20 (pseudocolour cyan) and the IMM/matrix marker PMPCB (pseudocolour magenta), in wild-type (WT) or Fmr1 KO female mice. TOMM20 and PMPCB were merged to show partial marker overlap. Dotted area: magnified region of interest (ROI) to illustrate membrane integrity loss in Fmr1 KO mice compared with wild-type. Arrows show TOMM20-positive, but PMPCB-negative mitochondria. The dotted arrow shows PMPCB-positive, but TOMM20-negative mitochondria. Scale bar: 10 µm. A.U.: arbitrary units.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_9994/pm40359994/pm40359994__page6_image1.jpg)
