Table 2.

Optimized staining protocol for HER2 expression

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

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.

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.