Immunohistochemical Analysis of Pulmonary and Splenic Inflammation

After METH administration and Ag sensitization, each mouse was euthanized and lung/spleen tissues were excised and fixed in 4% paraformaldehyde (Sigma) for 24 h. Tissues were processed, embedded in paraffin, and 4 μm sagittal sections were fixed to glass slides. The tissues were then subjected to hematoxylin and eosin (H&E) staining, CD4- (clone W3/25)^{44 (link)}, and CD8- (clone 32-M4)^{45 (link)} specific antibody (Ab; conjugated to horseradish peroxidase; dilution: 1:1000; Santa Cruz Biotechnology; SCB) immunostaining to assess tissue morphology, helper T cell, and cytotoxic T cell infiltration (brown), respectively. The slides were visualized using an Axiovert 40 CFL inverted microscope (Carl Zeiss; CZ), and images were captured with an AxioCam MRc digital camera using the Zen 2011 digital imaging software. The hyperplasia of type II pneumocytes was recorded microscopically in single cuboidal cells showing large nuclei, prominent nucleoli, and scant to abundant cytoplasm. The infiltration of CD4⁺ and CD8⁺ cells to pulmonary and splenic tissue was quantified by cell counts using the recorded 40 × images. Each image was blindly analyzed by three independent investigators. Ten microscopic fields per image were counted and the average was calculated per image (n = 10 images; 10 fields per image; 2 images per mouse; 5 mice per condition).

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Hernandez-Santini A.C., Mitha A.N., Chow D., Hamed M.F., Gucwa A.L., Vaval V, & Martinez L.R. (2021). Methamphetamine facilitates pulmonary and splenic tissue injury and reduces T cell infiltration in C57BL/6 mice after antigenic challenge. Scientific Reports, 11, 8207.

Publication 2021

paraformaldehyde Axiovert 40 CFL inverted microscope AxioCam MRc digital camera

Antibody Cd8 cells Cells Clone Cuboidal Cytoplasm Cytotoxic t cell Dilution Embedded paraffin Eosin Helper t cell Hematoxylin Horseradish peroxidase Hyperplasia Meth Mice Microscopic Nuclei Nucleoli Paraformaldehyde Pulmonary Splenic Tissue Type ii pneumocytes

Corresponding Organization :

Other organizations : University of Puerto Rico at Ponce, New York Institute of Technology, The University of Texas at El Paso, University of Florida, Florida College, Farmingdale State College, Long Island University

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Variable analysis

independent variables

METH administration
Ag sensitization

dependent variables

Tissue morphology
Helper T cell infiltration
Cytotoxic T cell infiltration
Hyperplasia of type II pneumocytes

control variables

Tissue fixation in 4% paraformaldehyde for 24 h
Tissue processing and embedding in paraffin
Sectioning at 4 μm
Hematoxylin and eosin (H&E) staining
CD4-specific antibody (clone W3/25) immunostaining
CD8-specific antibody (clone 32-M4) immunostaining
Microscopic visualization using Axiovert 40 CFL inverted microscope
Image capture using AxioCam MRc digital camera and Zen 2011 software
Blinded analysis by three independent investigators
Counting of 10 microscopic fields per image, with an average calculated per image (n = 10 images; 10 fields per image; 2 images per mouse; 5 mice per condition)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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