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

Marcos B. Salles, Bruno König, Sergio Allegrini, Marcelo Yoshimoto, Marília T. Martins, Paulo Coelho

Research output: Contribution to journalArticle

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 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 10-15μm 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.

Original languageEnglish (US)
Article number3307
JournalMedicina Oral, Patologia Oral y Cirugia Bucal
Volume16
Issue number1
DOIs
StatePublished - Jan 1 2011

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Ethanol
Formaldehyde
Polymers
Immunohistochemistry
Bone and Bones
Control Groups
Polymerization
Femur
Glycerol
Wistar Rats
Prostate
Microscopy
Anti-Idiotypic Antibodies
Joints
Alcohols
Carcinoma
Temperature
Proteins

Keywords

  • Immunohistochemistry
  • NF-kB
  • Nondecalcified sections
  • Technovit 7200
  • Wistar rats

ASJC Scopus subject areas

  • Dentistry(all)
  • Otorhinolaryngology
  • Surgery

Cite this

Identification of the nuclear factor kappa-beta (NF-kB) in cortical of mice Wistar using Technovit 7200 VCR®. / Salles, Marcos B.; König, Bruno; Allegrini, Sergio; Yoshimoto, Marcelo; Martins, Marília T.; Coelho, Paulo.

In: Medicina Oral, Patologia Oral y Cirugia Bucal, Vol. 16, No. 1, 3307, 01.01.2011.

Research output: Contribution to journalArticle

Salles, Marcos B. ; König, Bruno ; Allegrini, Sergio ; Yoshimoto, Marcelo ; Martins, Marília T. ; Coelho, Paulo. / Identification of the nuclear factor kappa-beta (NF-kB) in cortical of mice Wistar using Technovit 7200 VCR®. In: Medicina Oral, Patologia Oral y Cirugia Bucal. 2011 ; Vol. 16, No. 1.
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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{\circledR} 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 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 10-15μm 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.",
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AU - Yoshimoto, Marcelo

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AU - Coelho, Paulo

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AB - 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 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 10-15μm 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.

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