Immunofluorescence staining and analysis of cell‐assembled extracellular matrices

We performed immunofluorescence of specific proteins in the self‐assembled matrices using antibodies against individual ECM components. Matrices were fixed using 4% paraformaldehyde (PFA) for 20 min, followed by wash and blocking steps with 5% goat serum (Vector Laboratories, Burlingame, CA, U.S.A.) in PBS for 30 min. Primary antibodies were diluted in PBS and placed on matrices overnight at 4°C (Table ​(Table1).1). Secondary antibodies were used for 1 h at RT (Table ​(Table1).1). Finally, the matrices were washed three times in PBS and mounted on glass slides with Vectashield mounting medium (Vector Laboratories). After staining, ECMs were imaged using a Zeiss LSM‐510 inverted confocal microscope (Carl Zeiss, Jena, Germany). For each FN1‐stained sample, three random Z‐stack images of matrices were acquired; these were used for the evaluation of alignment and fibre measurements. The thickness of the ECMs was determined by taking Z‐stack images on the confocal microscope and subtracting the distance between the top and the bottom of assembled FN1 matrix. The images were subsequently processed with the ImageJ (National Institutes of Health, Bethesda, MD, U.S.A.) and Fiji (http://fiji.sc) programs. Each Z‐stack set of images was converted to a single image by using the maximum projection function. The ‘Dimensionality’ plug‐in was applied to calculate the orientation distribution of fibres, whereas the diameter of fibres and interfibrillar spaces were assessed with the ‘BoneJ’ plug‐in.56 All graphs were generated using GraphPad Prism version 6·01 (GraphPad Inc., La Jolla, CA, U.S.A.). Data are shown as the mean; error bars (±) are the SD of the mean. A one‐way anova followed by Bonferroni's correction was performed for experiments, with P < 0·05 considered significant.

Primary and secondary antibodies used in this study

^aSt Louis, MO, U.S.A.; ^bCambridge, U.K.; ^cEugene, OR, U.S.A.