Demonstration of progesterone receptors in paraffin wax sections of breast carcinoma.

Abstract

In the preliminary part of the study, paraffin wax sections of two progesterone receptor strongly positive tumours and one progesterone receptor negative tumour were used. Six sections from each case were investigated. Two were pretreated with trypsin, two with DNAse,2 and two were not pretreated. One section from each group was used for the demonstration of progesterone receptors by the peroxidase-antiperoxidase (PAP) method, and the other section was used for the demonstration of the receptor by the avidin-biotin-peroxidase complex (ABC) technique. The ABC technique, with no pretreatment, was the only method that gave good results that could be clearly visualised and was adopted for the rest of the investigation.In the next phase of the investigation 10 cases of breast carcinoma were studied. These included five progesterone receptor positive and five negative cases, assayed biochemically.Three paraffin wax sections from each case were studied. The receptors were investigated in two sections by the ABC technique, but the final staining result was developed in one by diaminobenzidine (DAB) and in the other by a mixture of DAB and imidazole.3 The third section was used as a negative control.Briefly, the sections were dewaxed by placing the slides in two changes of xylene, for two minutes each, and then hydrated through graded alcohols. Endogenous peroxidase activity was blocked by applying 3% hydrogen peroxide in methanol for 30 minutes. After rinsing in three changes for five minutes each of 0-2M TRIS-buffered-saline (TBS), pH 7-6, the sections were covered with normal goat serum for 30 minutes. This was then tipped off and the sections incubated with a few drops of PgR-ICA monoclonal antibody for 16-20 hours at 4°C. After rinsing in three changes of TBS for five minutes each sections were covered for two hours by biotinylated anti-rat IgG (Vector Laboratories), diluted 1/100 in TBS. Sections were then rinsed in TBS as before and incubated for two hours with avidin-biotin complex (Dakopatts). After rinsing in TBS, the sites of peroxidase activity were visualised by incubating the sections in 0-05% DAB (Sigma) and 0-01% hydrogen peroxide, either with or without added 0-05% imidazole, for six and 15 minutes, respec-

Immunohistochemical localisation of pS2 protein in ductal carcinoma in situ and benign lesions of the breast

“…For PR (Soomro & Shousha, 1990), paraffin sections prepared as for ER, were covered with normal goat serum for 30 min, then incubated with two drops of PR monoclonal antibody (Abbott Laboratories, UK) at 4°C overnight. After rinsing in 0.2 M Tris buffered saline, sections were incubated with biotinylated anti-rat IgG (1:100 dilution) for 2 h, then rinsed and incubated with avidin-biotin complex (Dako) for 2 h, followed by procedures described for ER, before counterstaining.…”

Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early-stage breast carcinomas: Prognostic significance

“…(-) <30 fmoUmg and (---) 230 fmoUmg. embedded tissues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]35,36]. The quantitative validity of these techniques has generally been established by comparison to ligand-binding assay results.…”

Even Tevye Knew Traditions Don't Last Forever

“…Shintaku et a1 used H222 antibody to demonstrate ER positivity in DNase-predigested formalin fixed, paraffin embedded tissues (ER-ICAP) (8). Other investigators demonstrated PR positivity in paraffin sections with KD68 without predigestion (PR-ICAP) (9). This technology, however, got mixed reviews; some investigators reported impressive correlation with the DCCA, whereas others obtained variable results (10).…”