Fig 1.
Fixation process prior to embedding muscle for cryosectioning visually and quantitatively preserves sarcomere structure.
(A) Representative images of fixed (top) and non-fixed (bottom) TA cryosections labeled with antibodies specific to α-actinin (rabbit polyclonal, Z-disc; red), myomesin (mouse monoclonal, M-line; green), and the titin-MIR domain (chicken polyclonal, sarcomere A/I-band interface; blue). Enhanced images are shown highlighted with yellow border. (B) Quantification of average sarcomere length between fixed and non-fixed TA sections. (C) Quantification of Z-disc width between fixed and non-fixed TA sections (D) Violin plots demonstrating distribution of values obtained from individual biological replicates. Quantifications are from 5 non-overlapping images acquired from n = 3 biological replicates per group. Solid bars represent median values, with dashed lines indicating upper and lower quartiles. Plotted measures in all figures are shown as mean of total measures with standard deviation, statistical comparisons shown are between group means, ***p<0.001, * p<0.05. Images obtained using confocal microscopy under 60x oil-immersion objective (See Methods).
Fig 2.
Comparison of sarcomere length homogeneity measures performed in this study to literature measures from in vivo imaging.
Measures of sarcomere length homogeneity from this study were plotted to compare to in vivo data reported in Moo et al. 2016. (A) Standard deviation (SD) of average sarcomere length measures. (B) Coefficient of variation (CV) of average sarcomere length measures. Data plotted is representative of the SD or CV obtained from the average sarcomere length measures obtained in this study (“Unfixed” or “Fixed (90°”), or of SD and CV values from “Deep Sarcomere” measures from Fig 5 in Moo et al., 2016 (“in vivo (50°)” or “in vivo (120°)”). Angles (°) in figure legend refer to the relative angle between foot and tibia used during measurements.
Fig 3.
Utilizing a FAB antibody post-blocking step prior to addition of subsequent same-host primary antibodies significantly limits potential overlap of secondary antibody labelings.
(A) Cartoon (produced with BioRender; not to scale) demonstrating an example of the labeling scheme used which directly corresponds to labeling sequence used in panel E. Black circles refer to the specific steps in the protocol outlined in Materials and Methods. (B) Representative image showing result of applying two same-host primary antibodies at the same time followed by simultaneous incubation with 488- and 647-conjugated secondary antibodies. (C) Representative image demonstrating application of two same-host primary antibodies, followed by incubation with 488- and 647-conjugated secondary antibodies, but with applications temporally segregated. (D) Representative image of two same-host primary antibody incubations including post-blocking steps using anti-rabbit unconjugated FAB antibodies prior to subsequent antibody incubations. (E) Representative image of TA section labeled triple-labeled with same-host primary antibodies corresponding to steps in panel A. Text above individual panels corresponds to specific antibody incubations (black circles with numbers in panel A) and/or wash steps (“wash”) used in the labeling protocol; sequential steps are separated by a comma, whereas simultaneous steps are grouped with a “+” (See Materials and methods for detailed protocol). Images obtained using SIM under 60x oil-immersion objective (See Materials and methods).
Fig 4.
Demonstration of a simple, reproducible approach to generate specified points of reference corresponding to fluorescently labeled proteins through image pre-processing in Fiji software.
(A) Example images of a TA cryosection labeled for an antibody specific to the N2A domain of titin with average background subtracted (left, in red), binarization applied (middle), and particles analyzed (right). (B) Example images with corresponding plot profiles demonstrating difference in pre- and post-processed images. (C) Demonstration of differences in points of reference for measures of sarcomere structure between pre- and post-processed images. (D) Comparison of sarcomere length analysis of identical line scans using either Fast Fourier Transform (F.T.) or optimized approach (Threshold/AP). * Student’s t-test p<0.05, # F-test p<0.0001 Images obtained using confocal microscopy using 63x oil-immersion objective (See Materials and methods for details).
Fig 5.
Representative images demonstrating increased nanoscale resolving capability using SIM compared to confocal microscopy.
(A) Representative image of TA cryosection labeled with antibody specific to the Titin N2A domain and imaged with confocal microscopy. (B) Representative image of TA cryosection labeled with antibody specific to the Titin N2A domain and imaged with SIM. Images obtained using either confocal microscopy under 63x oil-immersion objective or SIM under 60x oil-immersion objective (See Materials and methods).
Fig 6.
Utilization of fluorescently conjugated nanobodies enhances nanoscale protein localization accuracy of SIM.
(A,B,C) Representative images of TA cryosections labeled with an antibody specific to α-Actinin prior to incubation with (A,B) traditional IgG or (C) VHH secondary nanobodies and imaged using either (A) confocal microscopy or (B,C) SIM. (D) Quantification of Z-disc width measures obtained from confocal microscopy, SIM, and SIM + VHH secondary nanobodies. (E) Cartoon (not to scale, made with BioRender) depicting labeling scheme used for images in panel F. Numbers in black circles represent individual antibody incubations as in Materials and Methods. (F) Representative image demonstrating clear localization of three targeted primary antibodies (α-Actinin, Titin-N2A, Titin-PEVK) with nanobody secondaries within a nanoscale proximity. Images obtained using either confocal microscopy under 63x oil-immersion objective or SIM under 60x oil-immersion objective. Quantifications were performed using a minimum of n = 3 biological replicates per group, 5 non-overlapping images per biological replicate, and 5 non-overlapping line scans per image. Violin plots demonstrate the distribution of measures obtained per experimental group. Statistical comparisons were made using one-way ANOVA analysis, **** p<0.0001, *** p<0.001, * p<0.05.
Fig 7.
Cartoon demonstrating process of muscle fixation prior to preparation for longitudinal sectioning.
In brief, the mouse leg with the tibialis anterior muscle still attached is skinned and removed. The leg is placed on a piece of cork with knee and ankle joints fixed at standardized angles. The leg is then placed in 10% neutral-buffered formalin overnight. The following days, the muscle is removed, washed, and cryoprotected through a sucrose gradient. It is then frozen in OCT compound for cryosectioning. Cartoon generated using BioRender.See Materials and methods for details.
Fig 8.
Cartoon demonstrating an example labeling scheme with three same-host primary antibodies with corresponding sarcomere locations across a half-sarcomere.
Numbers in black circles correspond to the individual incubation steps listed. (1) α-Actinin antibody is used to label the sarcomere Z-disc. (2) AlexaFluor 647 secondary antibody is used to label α-Actinin antibody. (3) Anti-rabbit unconjugated FAB antibodies are used to block residual open binding sites on α-Actinin primary antibody. (4) Titin-MIR antibody is used to label the sarcomere A/I-band interface. (5) AlexaFluor 405 secondary antibody is used to label the Titin-MIR primary antibody. (6) Anti-rabbit unconjugated FAB antibodies are used to block residual open binding sites on Titin-MIR primary antibody. (7) Titin C-terminus antibody is used to label the sarcomere M-line, or middle of the sarcomere. (8) AlexaFluor 488 secondary antibody is used to label Titin C-terminus antibody. (9) Anti-rabbit unconjugated FAB antibodies are used to block residual open binding sites on Titin C-terminus primary antibody. Cartoon (not to scale) produced with BioRender.
Table 1.
Antibodies and concentrations used in the current study.