Figure 1.
Bodipy-493/503 versus Oil Red O.
Consecutive cryosections from human VL muscle were stained with ORO or Bodipy-493/503. Lambda stacks of the fluorescence emitted by Bodipy and ORO stained muscle sections were acquired with a LSM780, with a resolution of 9 nm (A). Emission spectra were graphed from the lambda stack and show that Bodipy’s emission spectra (B) is narrower than ORO’s (C). Furthermore, for both lipophilic dyes, the results show that lower emitted wave lengths seem to originate from stained intracellular membranes, while the higher wave lengths originate from the lipid droplets. (D) The detection of the emitted fluorescence from the Bodipy or ORO stained muscle cross-sections were combined with imaging of transmitted light through the cryosections, showing that transmission of light through the stained section is clearly blocked by the ORO stained lipid droplets, but unaltered by Bodipy stained lipid droplets (arrows).Bars; 5 µm.
Figure 2.
Optimization of human muscle lipid and glycogen stain.
Consecutive cryosections from 4 human VL muscle were either cut at −20°C and fixed right away or allowed to air dry for 15 or 30 min before staining of IMTG. (A) Representative high resolution images of individual muscle fibers are shown (bars; 20 µm). For each muscle (n = 4) and each condition 12 muscle fibers were used to quantify lipid droplet size and density. Lipid droplet density decreased 66 and 73% due to 15 or 30 min air drying, respectively. Furthermore, the size of lipid droplets decreased 37 and 43% due to 15 or 30 min air drying, respectively. (B) Air drying of muscle cryosections also has an effect on the preservation of intracellular structures, such as sarcoplasmic reticulum (SR) and mitochondria (mito). Bars, 15 µm. Arrows in the representative images of SR staining point to the areas that are magnified to visualize the ultra-structural alteration of SR during air drying. (C) Three protocols were compared to investigate the effect of freezing consecutive muscle cryosections at different time points during the staining protocol. In protocol 1 muscle cryosections were cut, stained and imaged the same day. In protocol 2 and 3, muscle cryosections were frozen and kept at −20°C for 3 weeks after or before Bodipy staining, respectively. Fiber typing was performed by immunostaining against myosin heavy chain I and II on a consecutive cryosection. Quantification of lipid droplet stain intensity and lipid droplet density was performed in 20 fibers from each muscle (n = 4) and, the results are plotted (D). The intensity of lipid droplet stain is expressed in arbitrary units (mean grey value), while the density of lipid droplets is expressed as number of lipid droplets per 3.6 µm2. The results show a clear loss of lipid droplet staining and density in protocols 2 and 3, compared to protocol 1. Statistical significance versus Protocol 1 is represented as *p<0.05, **p<0.01 and ***p<0.005 and statistical significance between type I and type II is represented as ¤p<0.05 (E) In order to optimize muscle glycogen preservation in muscle cryosections, three different fixatives were tested: Acidic ethanol, 4% PFA in ethanol and 4% PFA supplemented with 0.15% picric acid (Zamboni). Representative images of glycogen and laminin co-immunostaining of cryosections fixed with the three different fixatives are shown. Bars; 20 µm. A high resolution image of glycogen and laminin co-immunostaining showing preservation of glycogen particles in arterial smooth muscle is presented (F) to highlight the optimal preservation of glycogen when using 4% PFA supplemented with 0.15% picric acid. Smooth muscle fibers are smaller and more fragile than skeletal muscle single muscle fibers.
Figure 3.
Staining of human VL muscle lipid and glycogen stores with an optimized protocol reveals the existence of four metabolically distinct fiber types.
(A) Representative images of consecutive muscle crossections stained for lipid (top), glycogen and laminin (middle) and myosin heavy chain I (green) and II (red)(bottom) are presented. Bars; 20 µm. (B) ATPase staining of consecutive sections allowed for type IIa and IIx distinction. (C) Lipid (grey bars) and glycogen (black bars) stores were analyzed in vastus lateteralis muscle from 6 young healthy subjects and, the results are plotted as mean grey value ± SEM. Statistical differences versus I-1 are represented as *, versus I-2 are represented as # and, versus IIA are represented as ¤. Measurement of Mean Grey Value does not allow differentiating IMTG content in the 4 fiber types. A more detailed analysis of IMTG stores, as lipid droplets’ density and size (D), allows for the differentiation of 4 metabolically distinct fiber types. Two populations of type I muscle fibers, I-1 and I-2, can be differentiated by muscle fiber crossectional area (E) and lipid droplet density. Type I-2 muscle fibers are similar in fiber crossectional area and lipid content and distribution to type IIA. Quantification of lipid and glycogen content in 10 human VL muscles before (black bars) and after exhausting exercise (grey bars) was performed. Glycogen content is expressed as mean grey value (F), lipid droplets’ density as number of lipid droplets in 3.6 µm2 (G) and, lipid droplets’ size as number of pixels (H) (Pixel size is 3.6*10−3 µm2). Two-way repeated Measures Analysis of Variance was performed and statistically significant differences between fiber types (C and D: *vs I-1, #vs I-2) and, between basal and exhausted muscle (F–H: *p<0.05, **p<0.01 and ***p<0.005) are represented.