Protocols
BOND-MAX automated immunohistochemistry
Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas
Immunohistochemical staining was performed as follows: 2-μm-thick sections were prepared from formalin-fixed paraffin-embedded TMA tissue blocks and were dried in a 60°C oven overnight. The sections were placed in a BOND-MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH8.8) at 98°C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated with the primary antibody for 30 min. Subsequently, tissues were incubated with polymer for 10 min and developed with DAB-Chromogen for 10 min.
BOND-MAX Automated Immunohistochemistry Vision Biosystem
Standard 0/10 Protocol
1º Perosidase Block 10min
2º BOND Wash 0min
3º BOND Wash 0min
4º BOND Wash 0min
5º Marker 30min
6º BOND Wash 0min
7º BOND Wash 0min
8º BOND Wash 0min
9º Post Primary 10min
10º BOND Wash 2min
11º BOND Wash 2min
12º BOND Wash 2min
13º Polymer 10min
14º BOND Wash 2min
15º BOND Wash 2min
16º Distilled Water 2min
17º DAB 0min
18º DAB 10min
19º Distilled Water 0min
20º Distilled Water 0min
21º Distilled Water 0min
22º Distilled Water 0min
23º Hematoxilin 5min
24º Distilled Water 0min
25º BOND Wash 0min
26º Distilled Water 0min