Maa 2

To detect alpha 2,3-linked sialic acids we used the lectins Maackia amurensis agglutinin (MAA) in its two isoforms MAA I and MAA II (Vector Laboratories, USA). The MAA I lectin was FITC labeled. To detect alpha 2,6-linked sialic acids we used the lectin Sambucus nigra agglutinin (SNA) labeled with FITC (Vector Laboratories, USA). Cryosections of the different oviduct parts were incubated with the indicated lectins for 1 h at room temperature. MAA II was used after preincubation of sections with an Avidin/Biotin Blocking kit (from Vector Laboratories, USA). Detection of bound MAA II lectins was carried out with streptavidin labeled with Cy3 (Sigma-Aldrich).

The following biotinylated sialic acid-specific lectins were used to detect the avian and human influenza receptors [16 (link)]: biotinylated Maackia amurensis lectin I (MAA I); biotinylated Maackia amurensis lectin II (MAA II); and biotinylated Sambucus nigra agglutinin (SNA). MAA I (Cat# B-1315) and MAA II (Cat# B-1265) are markers for avian influenza virus receptors (AIV-R), while SNA (Cat# B-1305) is the marker for human influenza virus receptors (HuIV-R); the lectins were purchased from Vector Laboratories (Burlingame, CA, USA). Lectin histochemistry was performed, as described previously [17 (link)]. In brief, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, immersed in 3% hydrogen peroxide and incubated with 5% bovine serum albumin, to block nonspecific staining. The tissue sections were then allowed to incubate overnight with SNA (1.5 μg/mL) and MAA (6 μg/mL) in buffer at 4 °C. An SABC kit (Dako, Carpinteria, CA, USA) was used to optimize the contrast between specific labeling and background. Biotinylated lectin binding was revealed by a 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate-chromogen kit (Zymed Labs, South San Francisco, CA, USA), which produced a brown color, and the slides were counterstained with hematoxylin. Omission of the lectins was used as a negative control.

Human bronchus and lung samples were obtained from patients undergoing resection for lung tumors and archived formalin-fixed paraffin-embedded material was used. The collection of human respiratory tract tissues was approved by the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB) with written informed consent provided by study participants and/or their legal guardians. We used lung tissues from Caucasian (n = 10) and Asian (n = 20) patients. Tissue samples were obtained from formalin-fixed and paraffin-embedded tissues and stained for DBA lectin histochemistry and CT1 immunohistochemistry using methods below.
We used biotinylated SNA, MAA-I, and MAA-II from Vector Laboratories, and a biotinylated DBA from Vector Laboratories (DBA-I). Formalin-fixed and paraffin-embedded tissues were sampled, and slides were incubated with the above lectins at previously documented concentrations. Cad/Sda was detected using a monoclonal antibody provided by K. Klisch (University of Nottingham). Double staining using DBA and influenza nucleoprotein was used for ferret tissues that had been infected with pandemic H1N1 viruses and then euthanized (26 (link)).