Gs 700 imaging densitometer scanner

The molecular forms of MMP‐8 were detected by a modified enhanced chemiluminescence Western blot analysis kit according to the protocol recommended by the manufacturer (GE Healthcare) and described by Gürsoy et al. (2018 (link)) and Hanemaaijer et al. (1997 ). Briefly, the PISF samples were mixed with Laemmli sample buffer without any reducing reagents and heated for 5 min, followed by protein separation with 11% sodium dodecyl sulfate (SDS)‐polyacrylamide gels with prestained low range molecular weight sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) standards (Bio‐Rad) as molecular weight marker and human neutrophil (PMN) MMP‐8 (ProteaImmun) as a positive control. Target detections were performed by using a primary antibody, polyclonal anti‐MMP‐8, and a horseradish peroxidase‐linked secondary antibody (GE Healthcare). The immunoblots were quantified by GS‐700 Imaging Densitometer Scanner (Bio‐Rad) using the Quantity One program (Bio‐Rad).

The gelatinolytic activity was assayed from PISF samples with zymographic technique, using 11% SDS‐polyacrylamide gels impregnated with 1 mg/ml gelatin (Merck) as substrate, in line with previous descriptions by Paju et al. (2001 (link)) Sorsa et al.  (1997 ). Before the electrophoresis, the samples were incubated with Laemmli sample buffer without any reducing reagents for 2 h at room temperature. Prestained low range molecular weight SDS‐PAGE standards (Bio‐Rad) were used as molecular weight markers and human MMP‐9 (Merck) as a positive control. After electrophoresis, the gels were washed two times with 50 mM Tris‐HCl buffer and then incubated in 50 mM Tris‐HCl buffer, pH 7.5, containing 0.02% NaN3, 0.5 mM CaCl₂, and 1 μM ZnCl2 overnight at 370 C. The gelatinolytic activity was then visualized with 1% Coomassie Brilliant Blue R 250 solution; clear bands against the blue background on stained gels. The intensities of gelatinolytic activities were evaluated with GS‐700 Imaging Densitometer Scanner using Quantity One‐program (Bio‐Rad).