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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.