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Table 2 Optimized staining protocol for HER2 expression

From: A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation

Step

Reagent

Concentration

Interval

Manufacturer/Batch

1. Cell fixation

Methanol

1:1

5 min at RT

Merck KGaA, Germany, #I659409

2. Cell permeabilization

Dako Envision Target Retrieval solution™ (50×) (Tris/EDTA buffer solution, pH 9.0, and detergent)

1:50

20 min at 37°C

Dako Denmark, A/S, #20000821

3. AB serum

Dako AB diluent™ (1% FBS in PBS, 0.1% detergent)

1:1

20 min at RT

Dako Denmark, A/S, #00091216

4. HER2 labelling

Rabbit monoclonal AB specific to HER2

1:250

20 min at RT

Abcam plc, United Kingdom, #GR122507-5

5. ALP conjugation

ALP-conjugated porcine polyclonal anti-rabbit AB

1:50

30 min at RT

Dako Denmark, A/S, #20008362

6. LPR

Red chromogen and substrate buffert

1:100 (chromogen:substrate)

10 min at RT

Dako Denmark, A/S, #10082175

7. Nuclear counterstaining

Slowfade® Gold antifade with nuclear dye, DAPI

  

Life Technologies, United States, #1500156

  1. Washing with iced PBS 10.0% (v/v), 3 × 3 min between each step except after LPR when the slides are only quickly rinsed in with PBS before nuclear counterstaining.
  2. Abbreviations: AB, antibody; ALP, Alkaline phosphatase; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; EDTA, Ethylenediaminetetraacetic acid; LPR, liquid permanent red; PBS, phosphate buffered saline; RT, room temperature; Tris, 2-Amino-2-hydroxymethyl-propane-1,3-diol.