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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