笔记详情
标题
IHC Staining
内容
IHC Staining
Tissue microarray slides containing 285 formalin-fixed, paraffin-embedded breast cancer tissues were used to detect ERRF protein expression by IHC staining. In addition, 54 formalin-fixed, paraffin-embedded noncancerous breast tissues were used to monitor ERRF expression in noncancerous breast cancer tissues. Briefly, after deparaffinization in xylene and rehydration in a series of alcohols (100 − 75%), slides were incubated in the dual endogenous enzyme block (Dako, Carpinteria, CA) for 15 minutes to inactivate endogenous peroxide activity and were treated in citrate buffer (pH 6.0) for 3 minutes in a pressure cooker for antigen retrieval. After cooling for 45 minutes at room temperature, slides were incubated with rabbit anti-ERRF antibody (HPA026676; Sigma-Aldrich, St. Louis, MO) at 1:600 dilution at 4°C overnight and with the secondary horseradish peroxidase–labeled polymer anti-rabbit Igs (Dako) for 30 minutes at room temperature. With diaminobenzidine tetrahydrochloride (Dako) as a chromogen, slides were counterstained with hematoxylin. Preimmune serum was used as the negative control for ERRF antibody. The specificity of ERRF antibody was evaluated by IHC staining of T-47D (ERRF-positive) and MCF-7 (ERRF-negative) breast cancer cell lines prepared in paraffin blocks (see Supplemental Figure S1 at http://ajp.amjpathol.org).
Slides with IHC staining were examined independently by two investigators (X.F. and S.F.), and any discrepancy in the reading for a case was discussed and resolved. The intensity of nuclear staining (0 = negative, 1 = low, 2 = medium, and 3 = high) and the percentage of positively stained cells (0 − 100%) were recorded for each specimen, and ERRF expression was expressed as the multiplied score, which was calculated as intensity score × percentage for positive cells × 100. The average multiplied score for the 54 noncancerous breast tissues was ∼15. In addition, the median of ERRF expression levels in primary tumors was 15. Therefore, the cutoff point of 15 was used to classify primary tumors into two groups. Those with a multiplied score <15 were defined as lower ERRF expression, and those with a multiplied score ≥15 were defined as higher ERRF expression. We also used the receiver operating characteristic curve to determine the optimal cutoff points for ERRF expression. In this analysis, the optimal cutoff point is defined when the Youden Index, the potential effectiveness of a biomarker, achieved the maximum.21 With the Youden cutoff point, which varied among different variables, tumors were regrouped as ERRF higher or ERRF lower, and statistical analysis was performed again for each variable. 点击翻译
Tissue microarray slides containing 285 formalin-fixed, paraffin-embedded breast cancer tissues were used to detect ERRF protein expression by IHC staining. In addition, 54 formalin-fixed, paraffin-embedded noncancerous breast tissues were used to monitor ERRF expression in noncancerous breast cancer tissues. Briefly, after deparaffinization in xylene and rehydration in a series of alcohols (100 − 75%), slides were incubated in the dual endogenous enzyme block (Dako, Carpinteria, CA) for 15 minutes to inactivate endogenous peroxide activity and were treated in citrate buffer (pH 6.0) for 3 minutes in a pressure cooker for antigen retrieval. After cooling for 45 minutes at room temperature, slides were incubated with rabbit anti-ERRF antibody (HPA026676; Sigma-Aldrich, St. Louis, MO) at 1:600 dilution at 4°C overnight and with the secondary horseradish peroxidase–labeled polymer anti-rabbit Igs (Dako) for 30 minutes at room temperature. With diaminobenzidine tetrahydrochloride (Dako) as a chromogen, slides were counterstained with hematoxylin. Preimmune serum was used as the negative control for ERRF antibody. The specificity of ERRF antibody was evaluated by IHC staining of T-47D (ERRF-positive) and MCF-7 (ERRF-negative) breast cancer cell lines prepared in paraffin blocks (see Supplemental Figure S1 at http://ajp.amjpathol.org).
Slides with IHC staining were examined independently by two investigators (X.F. and S.F.), and any discrepancy in the reading for a case was discussed and resolved. The intensity of nuclear staining (0 = negative, 1 = low, 2 = medium, and 3 = high) and the percentage of positively stained cells (0 − 100%) were recorded for each specimen, and ERRF expression was expressed as the multiplied score, which was calculated as intensity score × percentage for positive cells × 100. The average multiplied score for the 54 noncancerous breast tissues was ∼15. In addition, the median of ERRF expression levels in primary tumors was 15. Therefore, the cutoff point of 15 was used to classify primary tumors into two groups. Those with a multiplied score <15 were defined as lower ERRF expression, and those with a multiplied score ≥15 were defined as higher ERRF expression. We also used the receiver operating characteristic curve to determine the optimal cutoff points for ERRF expression. In this analysis, the optimal cutoff point is defined when the Youden Index, the potential effectiveness of a biomarker, achieved the maximum.21 With the Youden cutoff point, which varied among different variables, tumors were regrouped as ERRF higher or ERRF lower, and statistical analysis was performed again for each variable. 点击翻译
来源
Am J Pathol. 2012 Mar; 180(3): 1189–1201.
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