Tissues were fixed in 10% formalin, embedded, and sectioned. The samples were fully deparaffinized in 100% xylene and rehydrated in ethanol at gradually lower concentrations. The slices were then soaked in citrate buffer (pH 6.0) and boiled in Retriever (Electron Microscopy Sciences, PA, USA) for antigen retrieval using standard procedures. For immunohistochemical (IHC) staining, the slices were stained with primary antibodies, followed by signal detection using a Signal Stain DAB Substrate Kit (Cell Signaling Technology, MA, USA). For immunofluorescence, the tissue samples or cells were fixed with 4% paraformaldehyde at room temperature and stained with primary antibodies, as described above. The general staining procedure was based on the standard protocols. Images were acquired using a BX53 microscope (Olympus, Japan).
Retriever
Manufactured by Electron Microscopy Sciences
Sourced in United States
The Retriever is a compact and versatile sample retrieval system designed for electron microscopy applications. It allows for the automated transfer of samples between multiple locations, such as storage, preparation, and imaging stations, to streamline the workflow. The core function of the Retriever is to facilitate the efficient and accurate handling of sensitive samples within the electron microscopy environment.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst, by Thermo Fisher Scientific (2 mentions)
R buffer a ph 6, by Electron Microscopy Sciences (2 mentions)
Anti il 8 antibody, by BD (2 mentions)
Lsm 700 upright laser scanning confocal microscope, by Zeiss (2 mentions)
Signalstain dab substrate kit, by Cell Signaling Technology (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Rabbit anti collagen 1, by Abcam (1 mentions)
Mouse anti cd68, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal anti cd3, by Agilent Technologies (1 mentions)
Mouse anti cd163, by Thermo Fisher Scientific (1 mentions)
Rat anti pnad, by BioLegend (1 mentions)
Goat anti lyve 1, by R&D Systems (1 mentions)
Dab substrate, by Thermo Fisher Scientific (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Bx53 light microscope, by Olympus (1 mentions)
Immpress hrp reagent, by Vector Laboratories (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
5 protocols using retriever
1
Immunohistochemical and Immunofluorescence Tissue Staining
2
Comprehensive Immunohistochemical Analysis of Lymph Nodes
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Immunohistochemistry was performed as previously indicated [73 (link), 82 (link)] on formalin-fixed paraffin-embedded (FFPE) LNs obtained at necropsy. Briefly, sections were deparaffinized and antigen retrieval was performed using a Retriever (Electron Microscopy Services, Hatfield, PA) in Tris-EDTA-Tween-80 buffer 73. Sections were stained for Tcells/B cells/dendritic cells (polyclonal rabbit anti-CD3, Dako, Santa Clara, CA; polyclonal rabbit anti-CD20, Thermo Fisher Scientific, Pittsburgh, PA; mouse-anti-CD11c, Leica Microsystems, Buffalo Grove, IL), macrophage subsets (mouse anti-CD68, Thermo Fisher; rabbit anti-DC-SIGN, ProSci Inc, Poway, CA; mouse anti-CD163, Thermo Fisher), LN vascular and structural aspects (Goat anti-LYVE-1, R&D Systems, Minneapolis, MN; rat-anti PNAd, BioLegend, San Diego, CA), and LN conduit systems (visualized by staining for rabbit anti-collagen 1 [Abcam, Cambridge, MA]). Primary antibodies were visualized with species- and isotype-specific secondary antibodies purchased from Jackson ImmunoResearch (West Grove, PA). Auramine rhodamine was performed as previously indicated [73 (link)] using reagents from BD Biosciences (San Jose, CA). Images were acquired at 20x magnification with a Nikon e1000 widefield microscope (Nikon, Melville, NY) with Nikon Elements.
3
Immunohistochemical Analysis of 3D Organoid Cultures
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Immunohistochemical techniques were carried out as previously described24 (link),25 (link). Paraffin sections of 3D organotypic cultures were baked for one hour at 60 °C and deparaffinized in xylene. Sections were then rehydrated in sequential 100%, 95%, 70% ethanol and distilled water. Antigen unmasking was performed with the Retriever (Electron Microscopy Sciences) in 10 mM sodium citrate buffer, pH 6. Endogenous peroxidases were quenched in 3% hydrogen peroxide for 6 min prior to sections being incubated for 30 min with a blocking solution of 5% BSA in PBS. Sections were incubated with primary antibodies overnight at 4 °C in 5% BSA, followed by 2 h with ImmPRESS HRP reagent (Vector Laboratories). HRP activity was then detected using DAB substrate (Thermo Scientific). Sections were dehydrated in 70%, 95%, and 100% ethanol and xylene, and coverslipped with Permount (Fisher Scientific). Sections were then imaged and photographed with an Olympus BX53 light microscope (Olympus America)24 (link),25 (link).
4
Immunofluorescent Staining of IL-8 in Lung Tissue
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Paraffin-embedded lung tissue blocks were baked on the hotplate at 75 °C for 20 min and deparaffinized in xylene. The slides were rehydrated from 100%, 90%, to 70% alcohol, and then to PBS. We performed the antigen unmasking using the retriever (Cat. # 62700–10, Electron Microscopy Sciences) with R-Buffer A pH 6.0 (Cat. # 62706–10, Electron Microscopy Sciences) for 2 h to complete the cycle and cool down. Slides were blocked with 20% normal goat serum (NGS) in PBST for 2 h at room temperature. Slides were incubated with an anti-IL-8 antibody (Cat. # 550419, BD Pharmingen™) in 5% NGS with PBS at 4°C for overnight. Slides were washed with PBST and incubated with Alexa 488 coated goat anti-mouse antibody in 5% NGS/PBS for 2 h at room temperature. Slides were washed with PBST and stained with Hoechst (1:5000 in PBS, Invitrogen). Coverslips were mounted on slides using ProLong Glass Antifade Mountant (Cat. # P36982, Invitrogen) and dried out in the dark overnight. Confocal images were acquired using the ZEISS LSM 700 Upright laser scanning confocal microscope and ZEN imaging software (ZEISS).
5
Immunofluorescence Staining of IL-8 in Lung Tissue
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Paraffin-embedded lung tissue blocks were baked on the hotplate at 75°C for 20 min and deparaffinized in xylene. The slides were rehydrated from 100%, 90%, to 70% ethanol and then to PBS. We performed the antigen unmasking using the retriever (Cat. # 62700-10, Electron Microscopy Sciences) with R-Buffer A pH 6.0 (Cat. # 62706-10, Electron Microscopy Sciences) for 2 h to complete the cycle and cool down. Slides were blocked with 20% normal goat serum (NGS) in PBST for 2 h at room temperature. Slides were incubated with an anti-IL-8 antibody (Cat. # 550419, BD Pharmingen™) in 5% NGS with PBS at 4°C overnight. Slides were washed with PBST and incubated with Alexa 488 coated goat anti-mouse antibody in 5% NGS/PBS for 2 h at room temperature. Slides were washed with PBST and stained with Hoechst (1:5000 in PBS, Invitrogen). Coverslips were mounted on slides using ProLong Glass Antifade Mountant (Cat. # P36982, Invitrogen) and dried out in the dark overnight. Confocal images were acquired using the ZEISS LSM 700 Upright laser scanning confocal microscope and ZEN imaging software (ZEISS).
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