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


METHODS
A 1.8 mm diameter defect was performed 0.5 mm 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 non-surgical 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°C for 7 days, and were dehydrated in a series of alcohols at -20°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°C. Polymerization followed the manufacturer's recommendation. The samples were grounded and polished to 10-15 m 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.


CONCLUSIONS
The results obtained support the feasibility of the developed immunohistochemistry technique.
Department of Anatomy, Institute of Biomedical Sciences, Universidade de São Paulo, Brazil.
Marcos B
MB
Salles
Bruno
B
König
Sergio
S
Allegrini
Marcelo
M
Yoshimoto
Marília T
MT
Martins
Paulo G
PG
Coelho
D000180:Acrylic Resins; D000818:Animals; D001842:Bone and Bones / Q000033:anatomy & histology*; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D016328:NF-kappa B; D051381:Rats; D017208:Rats, Wistar
D000180:Acrylic Resins; D016328:NF-kappa B; C053012:Technovit 7100