After rinsing three times with PBS for three min, citric acid antigen retrieval was performed under high pressure. The slices were blocked with normal goat serum for 30 min at room temperature to eliminate nonspecific staining, followed by incubation with primary antibody solution (Abcam, Cambridge, UK) at 4��C overnight. After recovery for selleckchem Brefeldin A 40 min at room temperature, ready-to-use secondary antibody solution (rabbit anti-mouse secondary antibody, Zhongshan Golden Bridge, Beijing, China) was added dropwise to the slices and incubated at room temperature for 40 min followed by three PBS washes for 3 min. DAB reagent was added dropwise to the slices afterwards and developed at room temperature.

Slices were observed under a microscope for three to five min to determine the optimal developing time, after which slices were rinsed with tap water, stained with hematoxylin for 90 seconds, differentiated with the hydrochloric acid solution, and treated with saturated aqueous lithium carbonate for blue nuclear staining. Slices were mounted with neutral gum after routine dehydration and observed under a microscope. A positive result was determined by evaluating both staining intensity and positive rates. If the positive rate was less than 10% with weak staining, it was labeled as negative; if the positive rate was greater or equal to 10% with strong brownish-yellow granules, it was labeled as positive. We also used reverse transcriptase polymerase chain reaction (RT-PCR) to detect NSE transcript levels in the bone marrow of patients.

Two ml bone marrow samples were extracted by bone marrow biopsy from previously untreated MM patients and control healthy subjects. Mononuclear cells were enriched by density gradient centrifugation. Trizol extraction of total RNA was performed followed by PCR (Takara DRR002B). The upstream primer sequence for NSE: 5��-GACTGAGGACACATTCATTGCTGAC-3��; downstream primer sequence: 5��-CAGCACACTGGGATTACGGAAG-3��. Eight ��l reaction product together with 2 ��l loading buffer was resolved on a 2% agarose ethidium bromide (EB)containing gel by electrophoresis. Results were documented under a UV transmission reflectometer. 3 Monitoring of patient condition indices Prior to each course of chemotherapy, weekly routine preoperative examinations were performed on each patient.

These included blood count, liver function, renal function, ��2-MG, serum NSE, serum protein electrophoresis, immunofixation electrophoresis, serum immunoglobulin (IgG, IgA and IgM) quantification, light chain (��,��) quantification, and bone marrow cell Entinostat morphology. Meanwhile MM-associated symptoms, such as bone destruction, infection, high viscosity syndrome, anemia, hypercalcemia, and renal damage, were monitored and recorded. 4 Statistical analysis SPSS16.0 software (Armonk, NY, USA) was used for statistical analysis.