Lung Tissue Immunohistochemistry for Key Markers

Five-micrometer thick, formalin-fixed, paraffin-embedded lung sections were immunohistochemically analyzed for the expression of HMGB1, forkhead box J1 (FOXJ1), club-cell secretory protein (CCSP), mucin 5AC (MUC5AC), mucin 5B (MUC5B), keratin 5 (KRT5), and KI-67. The sections were stained with the corresponding primary antibodies: rabbit polyclonal HMGB1 antibody (ab18256; Abcam, Cambridge, MA), rabbit monoclonal FOXJ1 antibody (ab235445; Abcam, Cambridge, MA), rabbit monoclonal uteroglobin antibody (ab213203; Abcam, Cambridge, MA), rabbit polyclonal MUC5AC antibody (UNC 294, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), rabbit polyclonal MUC5B antibody (UNC223, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), rabbit monoclonal Cytokeratin 5 antibody (ab52635; Abcam, Cambridge, MA), and rabbit monoclonal KI-67 antibody (ab16667; Abcam, Cambridge, MA), as published previously (34 (link), 35 (link)).

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Mao Y., Patial S, & Saini Y. (2023). Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease. Frontiers in Immunology, 13, 944772.

Publication 2022

ab18256 ab235445 ab213203 ab52635 ab16667

Antibodies Antibody Cell Cytokeratin 5 Formalin Hmgb1 Lung Monoclonal antibody Mucin 5ac Mucin 5b Paraffin Protein Rabbit Secretory Uteroglobin

Corresponding Organization : Louisiana State University

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Variable analysis

independent variables

Immunohistochemical staining for the expression of HMGB1, FOXJ1, CCSP, MUC5AC, MUC5B, KRT5, and KI-67

dependent variables

Expression levels of HMGB1, FOXJ1, CCSP, MUC5AC, MUC5B, KRT5, and KI-67

control variables

Five-micrometer thick, formalin-fixed, paraffin-embedded lung sections

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

Based on most similar protocols

Fixation of mouse lungs in 4% paraformaldehyde at 4°C overnight can be used to preserve tissue structure and antigenicity for immunostaining (Protocol 2, Protocol 4).

Antigen retrieval using high pH (9.0) antigen unmasking solution or proteinase K treatment can improve antibody binding and signal detection (Protocol 2, Protocol 3, Protocol 5).

Embedding lung tissue in paraffin blocks and sectioning at 3-5 μm thickness is a common approach to prepare samples for immunostaining (Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Using a range of primary antibody dilutions (e.g., 1:200-1:500) can help optimize signal-to-noise ratio and specificity (Protocol 2, Protocol 4).

Blocking with 3% bovine serum albumin (BSA) and 0.4% Triton X-100 in Tris-buffered saline (TBS) can help reduce non-specific antibody binding (Protocol 3, Protocol 5).

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