Left panel: epidermal layer of the root shown polar PIN2 localization. 
Right panel: The PIN2 signals were restricted only to cortex, epidermis and root caps. 

Material: Arabidopsist thaliana young seedling-5-day-old roots.
Fixation: 2 % Formaldehyde in Microtuble-stabilizing buffer stock solution (50 mM PIPES, 5 mM EGTA, 2 mM MgSO₄, 0.4 % Triton), vacuum for 3 min. and fixation conducted for 40 min. 
Washing buffer: Microtuble-stabilizing buffer stock solution (50 mM PIPES, 5 mM EGTA, 2 mM MgSO₄, 0.4 % Triton)
Primary antibody: 1: 200
Secondary antibody: anti-rabbit (1:250): (Rhodamine Red™-X (RRX) AffiniPure Donkey Anti-Rabbit IgG (H+L)/Jackson ImmunoResearch/ Cat# 711-295-152) 

And the unique signal pattern colocalized with PIN2-GFP marker line (in green). The PIN2 signal is not visible in the middle of a root tissue, where PIN2 protein does not occur (negative control). 



Material: Arabidopsist thaliana young seedling-5-day-old roots.
Fixation: 4 % Formaldehyde in 1×Phosphate buffered saline (PBS) (4g NaCl, 0.115g NaH2PO4•H2O, 0.695g Na2HPO4•2H2O, 0.1g KCl in 1L ddH2O, pH 7.4) supplement with 0.1% TritonX, vacuum for 1 hour, and fixation conducted for 1 hour.
Washing buffer: 1×Phosphate buffered saline (PBS)
Blcoking buffer: 3% BSA (Carl Roth, cat. No. 8076.3) in 1×PBS
Primary antibody: 1: 500 ON/4ºC
Secondary antibody: anti-rabbit (1:500), Alexa Fluor 568 (Invitrogen, A11011).

Courtesy SHEN Jinbo Zhejiang Agricultural & Forestry University, Hangzhou, Zhejiang, China