Fuchsin substrate chromogen

The isolation of the human primary osteoblasts (hOB) was performed according to a previously described protocol under sterile conditions [20 (link)]. The human primary osteoblasts were taken from the spongiosa of the femoral heads of patients who underwent total hip replacement. The study was approved by the Local Ethical Committee of Rostock, Germany (registration number: A2010-10), and an informed consent was signed by each patient.
The human osteoblasts were cultivated in modified Eagle’s osteogenic cell culture medium (MEM; Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% amphotericin B, and 1% HEPES buffer (all from Gibco-Invitrogen, Darmstadt, Germany) without calcium, and the osteogenic additives dexamethasone (100 mM), L-ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany). The osteogenic differentiation of the human primary osteoblasts was confirmed by immunohistochemical detection of the enzyme alkaline phosphatase using a fuchsin+substrate chromogen (DAKO, Hamburg, Germany). For the further tests, the isolated cells were cultured in 25-cm² flasks with 8 ml of osteoblasts in the same medium but without the 1% penicillin/streptomycin and under standard cell culture conditions (5% CO₂ and 37°C).

Isolation of human primary osteoblasts followed the procedure described previously by Lochner et al. [38 (link)]. Briefly, cells were isolated from the spongiosa of the femoral heads of patients undergoing primary total hip replacement. The samples were collected with patient agreement and approval by the Local Ethical Committee (registration number: A 2010-10). Cultivation was done in osteogenic cell culture medium (MEM Dulbecco, Biochrom AG, Berlin, Germany) with 10% FCS, 1% penicillin/streptomycin, 1% amphotericin B, 1% HEPES buffer (all: Gibco^®-Invitrogen, Darmstadt, Germany) including osteogenic additives (dexamethason (100 mM), L-ascorbic acid (50 mg/mL) and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany)). Alkaline phosphatase staining with fuchsin substrate chromogen (DAKO, Hamburg, Germany) was done to verify the osteogenic character of the isolated cells. Cultivation was carried out in an incubator (Binder GmbH, Tuttlingen, Germany) at simulated in vivo conditions 37 °C, 5% CO₂ and 21% O₂ for one week.
A total of seventeen different donors (10 ♂, 7 ♀) were used for all experiments. The average age was 66 ± 9.4 years. Cells were not pooled; one donor was used for one scaffold per experiment.

Sections obtained from formalin-fixed paraffin-embedded tissues were used for IHC. Following deparaffinization and antigen retrieval in Tris-EDTA buffer (pH 9.0), the specimens were incubated with primary antibodies, followed by incubation with the HRP- or AP-conjugated secondary antibodies, then the samples were visualized using the AEC plus substrate-chromogen (Dako), Fuchsin+ Substrate-Chromogen (Dako) and Histogreen Substrate kit for peroxidase (Abcys). The slides were counterstained with hematoxylin solution.