A Simplified Method for the Aspiration of Bone Marrow from Patients Undergoing Hip and Knee Joint Replacement for Isolating Mesenchymal Stem Cells and In Vitro Chondrogenesis

Table 2

List of antibodies used for IHC of micromass cartilages.

	Primary antibody	Secondary antibody
Type II collagen (COL II)	Mouse anti-COL II, monoclonal, II-II6B3, DSHB	Biotinylated horse anti-mouse IgG, rat adsorbed, BA-2001, Vector
Type X collagen (COL X)	Mouse anti-COL X, monoclonal, 2031501001, Quartett, GE	The same as above
Type I collagen (COL I)	Mouse anti-COL II, C2456, Sigma-Aldrich, ascites fluid	The same as above
Aggrecan	Rabbit polyclonal to aggrecan, ab36861, abcam	Biotinylated horse anti-rabbit IgG, BA-1100, Vector
Type VI collagen (COL VI)	Rabbit polyclonal to COL VI (Biotin), ab6583, abcam	None

(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).