Table 2: List of antibodies used for IHC of micromass cartilages.

Primary antibodySecondary antibody

Type II collagen (COL II)Mouse anti-COL II, monoclonal, II-II6B3, DSHBBiotinylated horse anti-mouse IgG, rat adsorbed, BA-2001, Vector

Type X collagen (COL X)Mouse anti-COL X, monoclonal, 2031501001, Quartett, GEThe same as above

Type I collagen (COL I)Mouse anti-COL II, C2456, Sigma-Aldrich, ascites fluidThe same as above

AggrecanRabbit polyclonal to aggrecan, ab36861, abcamBiotinylated horse anti-rabbit IgG, BA-1100, Vector

Type VI collagen (COL VI)Rabbit polyclonal to COL VI (Biotin), ab6583, abcamNone

(i) Primary and secondary antibodies were diluted in blocking buffer.
(ii) Negative control was without primary antibody and with mouse IgG (for types II, I, and X) and with rabbit IgG, polyclonal (ab27478, abcam), for aggrecan and COL VI.
(iii) For antigen retrieval for COLs I, II, X and aggrecan, the hydrated tissue sections were treated with pepsin (P-7000, Sigma-Aldrich; 4 mg/mL in 0.01 N HCl) for 10 min at 37°C, washed with dH2O (4x, 1 min), and then treated with hyaluronidase (H-3506, Sigma-Aldrich) solution at 1 mg/mL (in 0.1 M phosphate buffer, pH 5.0) for 30 min at 37°C and washed with dH2O (4x, 1 min).
(iv) For antigen retrieval for COL VI, hydrated sections were treated with 20 μg/mL proteinase K (EO0491, Thermo Fisher) for 15 min at 37°C.
(v) Blocking buffer constituted of PBS-T containing 2% BSA (ALB-001, albumin-bovine serum fraction V, Bioshop) and 2% horse serum (16050122, GIBCO).
(vi) PBS-T was composed of D-PBS containing 0.05% Tween-20 (TWN510, Bioshop).