Ab82968

IHC was performed to evaluate the expression of TGFBR2 and IGF2BP1 in 16 leiomyoma tissues. Tissues were fixed in formalin and embedded in paraffin, and 3-μm-thick paraffin sections were obtained. IHC was performed using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer’s protocol. The sections were deparaffinized and pretreated with a cell-conditioning solution (CC1, Ventana), followed by UV irradiation to abrogate the endogenous hydroperoxidase activity. The primary antibodies were diluted in Dako antibody diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and used under the following dilutions: TGFBR2 (1:100, ab61213, Abcam, Franklin Lakes, NJ, USA) and IGF2BP1 (1:100, ab82968, Abcam,). The sections were incubated with primary antibodies at room temperature for 32 min, and hybridized with HRP-conjugated secondary antibody (Ventana) for 8 min. The reaction was developed with diaminobenzidine (DAB; Dako) for 5 min and the slides were counterstained with hematoxylin II (Ventana) for 4 min and bluing reagent (Ventana) for 4 min. The sections were observed under light microscope (BX50, Olympus, Japan).

For immunohistochemical staining, deciduous and permanent teeth were fixed in 10% buffered formalin for 1 day, decalcified with 10% EDTA (pH 7.4; Fisher Scientific, TX, USA) for 8 weeks, embedded in paraffin, and then sectioned at a thickness of 3 µm. Specimens were subjected to IHC staining with antihuman IGF2BP1 (Ab82968, Abcam, Cambridge, UK; rabbit polyclonal, diluted 1∶100), antihuman CALB1 (Ab25085, Abcam; rabbit polyclonal, diluted 1∶400), LGR5 (Ab75732, Abcam; rabbit polyclonal, diluted 1∶50), and gamma-aminobutyric acid (GABA) A receptor, beta 1 (GABRB1; Ab51123, Abcam; rabbit polyclonal, diluted 1∶200). Endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide. Sections were incubated in 5% bovine serum albumin to block nonspecific binding. The primary antibodies were diluted to give optimal staining and the sections were incubated overnight. After incubation, EnVision + System-HRP Labeled Polymer Anti-rabbit (K4003, Dako North America, CA, USA; ready to use) was applied for 20 min. Color development was performed using 3,3′-diaminobenzidine substrate (Dako) and counterstained with Gill's hematoxylin solution (Merck, Darmstadt, Bermany). Negative control sections were treated in the same manner but without primary antibodies.

Total cellular protein samples were separated with 10% SDS PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies against E-cadherin (ab40772), N-cadherin (ab76057), Vimentin (ab8978, Abcam), IGF2BP1 (ab82968, Abcam), YY1 (ab109228, Abcam), CDK4 (ab199728, Abcam), cyclin D1 (ab16663, Abcam), CDK2 (ab32147, Abcam) and GAPDH (ab9485, Abcam), along with HRP-conjugated secondary antibody were obtained and employed. GAPDH served as internal control.