DIFFERENTIAL INTERFERENCE CONTRAST (DIC) & IMMUNOFLUORESCENCE (IF) MICROSCOPY

Morphological analysis in C2C12 mouse myoblast cells and at different stages of myogenesis. Panels A-E were viewed at a 600X total magnification with oil immersion and obtained by the DIC/DAPI overlay using Olympus IX51 inverted microscope and Cellsens software. The cells were cultured in media containing DMEM, 10% FBS, and 0.1% Penicillin/Streptomycin over 10 days. DIC microscopy techniques were performed and placed in overlay with DAPI immunofluorescence (nuclei; blue). Fixation took place at (A) the 80% confluent myoblast stage; (B; black closed arrowheads) day 0 myoblasts in cell-to-cell contact stage; (C) day 4 myotubes in early development; (D; white arrow head) day 7 myotubes in later myotube development; (E) day 10 mature myotubes.

*Note. Figure 1 images were taken with inconsistent gain and exposure levels. Contrary to immunofluorescent imaging, the DAPI staining is not imaged for experimental purposes in the DIC/DAPI overlays. They were adjusted accordingly to produce the best possible images to examine the morphology and nucleation of the cells.

Star-shaped mononucleated myoblasts are seen reaching out to nearby cells at the myoblast stage (A) and in fusion at the day 0 stage of differentiation (B). Myotubes appear increasingly thicker with more nuclei fusion continues, and they process from early (C) to later stages of development (D). Myotubes that are further in development appear thinner/elongated, therefore "squishing" the nuclei and develop their contractile ability (E).

Preliminary evidence shows that myoblasts are in early stages of myogenesis appear star-shaped and mononucleated (A) and they fuse together (B) to form thinner myotubes with more nuclei as myogenesis progresses (C-E).

The results align with that preliminary evidence.

IMMUNOCYTOCHEMISTRY (ICC) & IMMUNOFLUORESCENCE (IF) MICROSCOPY

Expression and localization of alpha- and beta-actin in C2C12 murine mouse myoblast cells at the myoblast stage and at different stages of myogenesis. All panels were focused at a 600X total magnification and obtained by the Olympus IX51 inverted microscope and Cellsens software. Alpha-actin was labelled in red, beta-actin was labelled in green, and the nuclei was labelled in blue. Fixation took place at (A) the 80% confluent myoblast stage; (B) day 0 at myoblasts cell-to-cell contact stage; (C) day 4 of early myotube development; (D) day 7 of later myotube development; (E) day 10 of mature myotubes; and (F) day 6 of negative control myotubes.

The immunofluorescence expression of beta-actin is prominent in A-C and localized at the throughout cell's cytoskeleton. The immunofluorescence expression of beta-actin is more diminished D-E when alpha-actin is predominant.

Preliminary results suggest that beta-actin immunofluorescence expression is more predominant at the cytoskeleton in early stages of myogenesis, for cell growth and motility, and declines throughout differentiation. Additionally, alpha-actin is more predominant in later stages of myogenesis, and is localized along the myotubes, when sarcomere are developing.

These results align with the preliminary data and our expected results that beta-actin will be more present than alpha-actin in early stages of myogenesis and beta-actin will be less present than alpha actin in later stages of myogenesis, due to their known roles in myogenesis.

*Note: The images were not taken at consistent settings, therefore, the immunofluorescence expression of beta-actin and alpha-actin cannot be directly compared. The images do, however, show a visual for what alpha-and beta-actin might look like at each sample day.

Expression and localization of alpha- and beta-actin in C2C12 mouse myoblast cells at the myoblast stage and at different stages of myogenesis. Panels Myoblasts and Day 0-10 in series a-d were focused at a 600X total magnification and obtained by the Olympus IX51 inverted microscope, Olympus BX53 upright microscope, and Cellsens software. TRITC (886.4 ms exposure time and 11.1 dB gain), FITC (886.4 ms exposure time and 8.1 dB gain), and DAPI (193.7ms exposure time and 2.9 dB gain) channel settings were consistent across all panels. The cells were cultured in media containing DMEM, 10% FBS, and 1% Penicillin/Streptomycin over 11-12 days. Alpha-actin was labelled in red (TRITC), beta-actin was labelled in green (FITC), and the nuclei was labelled in blue (DAPI). Fixation took place at (MB) the 80% confluent myoblast stage; day 0 (D0) at myoblasts cell-to-cell contact stage; day 4 (D4) of early myotube development; day 7 (D7) of later myotube development; day 10 (D10) of mature myotubes; and day 6 (D6) of myotube development (negative control with no primary antibodies).

*Note: Series a-d represent images taken at different days, over an increasing period of time since plating, hence why they are more diminished.

This figure depicts a more accurate representation of the immunofluorescence expression of myogenesis.

Beta-actin is predominantly expressed at MB and D0 throughout the cell's cytoskeleton and alpha-actin is more evident at D4, D7, and D10 along the myotube.

This again aligns with results from the preliminary data on beta-actin and alpha-actin immunofluorescence expression and localization that was previously mentioned.

Note: All images were taken in the Fall semester of 2021.