Fluorescence images were obtained using a confocal laser-scanning microscope (FV1000; Olympus). Scanning was performed in sequential laser emission mode to avoid scanning at other wavelengths. Images obtained from BM sections were analyzed using the TissueQuest image analysis software (TissueGnostics).
Tissuequest image analysis software
Manufactured by TissueGnostics
Sourced in United States
TissueQuest image analysis software is a digital image processing tool designed for the analysis of microscopic images. It provides a set of advanced algorithms and features for the quantification and characterization of cellular and tissue-level structures.
Automatically generated - may contain errors
Lab products found in correlation
Tissuefaxs whole slide scanning system, by TissueGnostics (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Fv1000, by Olympus (1 mentions)
Ab237728, by Abcam (1 mentions)
Ab254415, by Abcam (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab241332, by Abcam (1 mentions)
Tissuefax, by TissueGnostics (1 mentions)
Ld plan neofluar 20 0.4 objective, by Zeiss (1 mentions)
Tissuefaxs imaging software, by TissueGnostics (1 mentions)
5 protocols using tissuequest image analysis software
1
Confocal Imaging and Analysis
2
Immunofluorescence Analysis of Sinonasal Immune Markers
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Paranasal sinus tissues of the participants were embedded in paraffin and prepared into 4 μm thick sections. After paraffin removal, the deparaffinized sections were heat-treated in citrate repair buffer (pH 6.0), followed by the addition of drops of 3% hydrogen peroxide to remove endogenous peroxidase. The sections were treated with goat serum blocking solution for 20 minutes and then reacted with the following primary antibodies for 1 hour for immunofluorescence staining: CD3 (1:1500, ab16669, Abcam), CXCR5 (1:5000, ab254415, Abcam), TIM-3 (1:1000, ab241332, Abcam), PD-1 (1:500, ab237728, Abcam). After washing, the sections were incubated with a secondary antibody (PV-6001, ZSGB-BIO) for 20 minutes. Subsequently, a rapid reaction buffer (1:100, FFBN45, DMK) was added and the sections were incubated at room temperature for 5 minutes for color development. The antibody complexes were removed by microwave treatment and the markers were counterstained. The sections were counterstained with 4′,6-diamidino-2-phenylindole at room temperature for 5 minutes. The sections were finally closed with the antifade mounting medium. After completion of staining, the sections were imaged using the TissueFAXS whole-slide scanning system (TissueGnostics, USA) and analyzed using the TissueQuest image analysis software (TissueGnostics, USA) to obtain accurate cell counts and densities.
3
Tissue Immunostaining and Whole-Slide Imaging
4
Quadruple Immunofluorescence Staining of Skin
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Quadruple immunofluorescence stainings of skin cryosections were performed with directly and indirectly labeled monoclonal antibodies (Supplementary Table S3). In brief, after incubation with the primary antibodies overnight, an appropriate secondary fluorescence-labeled antibody was applied for 30 minutes at room temperature, followed counterstaining with DAPI. Immunostainings were controlled with isotype-matched conjugates (Supplementary Figure S5d). For evaluation of immunofluorescence results, images were acquired at room temperature using a Z1 Axio Observer microscope equipped with an LD Plan-Neofluar Â20/0.4 objective (Zeiss, Oberkochen, Germany) and quantified using TissueFAXS and/or TissueQUEST image analysis software (TissueGnostics, Vienna, Austria).
5
Quadruple Immunofluorescence Staining Protocol
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Quadruple immunofluorescence staining of the acetone-fixed cryosections was performed using directly labeled monoclonal and secondary antibodies for increased signal strength (Table S3 , Supplementary Materials ). Briefly, after rehydration and blocking, the slides were incubated in several steps with primary antibodies overnight at 4 °C and appropriate secondary antibodies for 30 min at room temperature, followed by counterstaining with DAPI. Appropriate isotype controls were stained at the same time. For evaluating the immunofluorescence results, the images were acquired at room temperature using a Z1 Axio Observer microscope equipped with an LD Plan-Neofluar ×20/0.4 objective (Zeiss, Oberkochen, Germany) using TissueFAXS imaging software and quantified with TissueQUEST image analysis software (TissueGnostics, Vienna, Austria).
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