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Cells were then fixed with ice-cold methanol and stained (Hema 3 Stain System).
Boyden chambers 8 µm were from Transwell-Costar Corp. Hema 3 Stain System was from Fisher Diagnostics.
Dako's Cytomation Envision double stain system (K5361) (Dako Ltd., Cambridgeshire, UK), was used performed according to the manufacturer's instructions.
Primary antibodies were detected by the Immunoperoxidase method using the IDetect Super Stain System HRP (ID labs Biotechnology).
For cleaved caspase 3 and neuronal nuclear antigen (NeuN) double labeling, the DAKO Envision G2 Double Stain System was used.
To further analyze the cellular localization of EGFR expression in type I and type II alveolar epithelial cells double immunohistochemistry analysis for EGFR and SP-A SFTPA1-Abbiotec-6F10 SFTPA1-Abbiotec-6F10 SFTPA1-Abbiotec-6F10wash slight modifications undertakenision double stasn system protocol for paraffin-embedescribedue sections.
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All procedures were done on the automated Ventana Discovery Ultra staining system.
IHC was performed using an automated staining system with antibodies against Kif4A (GTX115759, dilution 1 200; Gene Tex, USA), CD68 (lone KP1, dilution 1 100; ZM-006, ZSGB-bio, Beijng, China).
Immunohistochemistry was performed on de-paraffinized and rehydrated sections without pretreatment using the 3,3′-diaminobenzidine method on an automated staining system (Auto Stainer Link 48, DAKO, Glostrup, Denmark) according to the manufacturer's protocol.
After hematoxylin/eosin staining, the slides were stained with CD34 antibody (1 30, NCL-L END, Novocastra, Leica Biosystems, Nussloch, Germany) using an automated staining system (BenchMark XT, Ventana Medical Systems, Oro Valley, AZ, USA).
The staining procedure followed the manufacturer's instructions for the ABC staining system (Santa-Cruz Biotechnology).
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