Identification of the nuclear factor kappa-beta (NF-kB) in cortical of mice Wistar using Technovit 7200 VCR

Abstract

Objective: this study aimed to develop a nondecalcified bone sample processing technique enabling immunohistochemical labeling of proteins by kappa-beta nuclear factor (NF-kB) utilizing the Technovit 7200 VCR ® in adult male Wistar rats. Study Method: A 1.8 mm diameter defect was performed 0.5mm from the femur proximal joint by means of a round bur. Experimental groups were divided according to fixing solution prior to histologic processing: Group 1-ethanol 70%; Group 2-10% buffered formalin; and Group 3-Glycerol diluted in 70% ethanol at a 70/30 ratio + 10% buffered formalin. The post-surgical periods ranged from 01 to 24 hours. Control groups included a nonsurgical procedure group (NSPG) and surgical procedures where bone exposure was performed (SPBE) without drilling. Prostate carcinoma was the positive control (PC) and samples subjected to incomplete immunohistochemistry protocol were the negative control (NC). Following euthanization, all samples were kept at 4 o C for 7 days, and were dehydrated in a series of alcohols at -20 o C. The polymer embedding procedure was performed at ethanol/polymer ratios of 70%-30%, 50%-50%, 30%-70%, 100%, and 100% for 72 hours at -20 o C. Polymerization followed the manufacturer's recommendation. The samples were grounded and polished to 10-15μm thickness, and were deacrylated. The sections were rehydrated and were submitted to the primary polyclonal antibody anti-NF-kB on a 1:75 dilution for 12 hours at room temperature. Results: Microscopy showed that the Group 2 presented positive reaction to NF-kB, diffuse reactions for NSPG and SPBE, and no reaction for the NC group. Conclusion: The results obtained support the feasibility of the developed immunohistochemistry technique.

Amorphous calcium phosphate (ACP) in tissue repair process

“…For these samples, as against staining, Mayers' hematoxylin (EnVision tm Flex, Dako), was used. The analysis was performed, under standard light microscopy (Salles et al, ).…”

Evaluating Nuclear Factor NF-κB Activation following Bone Trauma: A Pilot Study in a Wistar Rats Model

“…Sixty-five Wistar male rats weighing ~350 g were divided into 13 groups ( n = 5 per group). The eight experimental groups (expG 1–8) were divided in accordance with the euthanasia time: zero, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h; the five control groups sham (conG 1–5) or false-operated groups were killed at zero, 1 h, 2h, 4 h, and 6 h. The sham group was performed only in these 5 times, because in a previous study performed and published to evaluate the technique used was found in the other time there was no mark [ 20 ], decreasing the number of animals used.…”

“…The samples were immersed in a fixative solution of 10% buffered formalin (0.1 molar phosphate buffer, pH 7.4), at 4°C for 7 days [ 20 ].…”

“…After the polymerization conducted using a conventional process, the samples were placed into polyethylene and resin (Techonovit ® 7200 VLC) clusters at – 20°C and covered completely. The material was subjected to light polymerization for a period of 9 h under white light and an additional 90 h under blue light [ 20 ].…”

“…Recently, an immunohistochemistry methodology was developed to analyse demineralization of the osseous cortex that aimed to expose the peak activation of NF-κB. The methodology not only indicated that the transcriptional factor was activated throughout the process of osseous cortex propagation but also demonstrated an increase in positive immune reactivity to this transcriptional factor in the knee joints in all studied groups [ 20 ]. This result indicates a possible joint degeneration mechanism in the knees (i.e., arthritis or arthrosis) due to the fact that the transcriptional factor is directly related to the stimulation of pro-inflammatory cytokines and osteoclastogenesis [ 21 – 23 ].…”