Skip to main content

Advertisement

Table 1 Optimized staining protocol for ERα, CK, and CD45 expression in CTCs and leukocytes

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™ (50x) (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 1 . ERα labelling Rabbit monoclonal AB specific to ERα 1:50 60 min at 37°C Thermo Scientific, United States, #9101S1210D
5 1 . CD45 staining Alexa Fluor 647 labeled mouse monoclonal AB specific to CD45 1:5 60 min at 37°C AbD Serotec, UK, #B173123
6 2 . ERα staining Alexa Fluor 488 labeled goat anti-rabbit secondary AB 1:200 45 min at RT Life Technologies, United States, #1423009
7 2 . Cytokeratin 8, 18, and 19 staining Phycoerythrin labeled mouse monoclonal AB, specific to CK 8, 18, and 19 0.0006% 45 min at RT Janssen Diagnostics, United States, #E491A
8. 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.
  2. Abbreviations: AB - antibody, DAPI - 4′,6-diamidino-2-phenylindole dihydrochloride, EDTA - Ethylenediaminetetraacetic acid, ERα - estrogen receptorα, PBS - phosphate buffered saline, RT - room temperature, Tris - 2-Amino-2-hydroxymethyl-propane-1,3-diol.
  3. 1 Step 4 and 5 can be performed simultaneously.
  4. 2 Step 6 and 7 can be performed simultaneously.