Fresh pancreatic or subcutaneous tumors were rinsed in PBS and placed in Zinc Formalin Fixative (Sigma-Aldrich) overnight. Then they were dehydrated serially in 70%, 95%, and 100% ethanol. The tissues were then embedded in paraffin, which were processed to generate H&E and unstained sections. To rehydrate for immunofluorescence, unstained sections were serially submerged in Xylene (Sigma), 100% ethanol, 95% ethanol, 70% ethanol, and PBS. Sections were then blocked with 10% donkey serum + 0.1% TritonX-100 in PBS overnight at 4°C; followed by incubation with a goat anti-GFP antibody (Abcam, ab6673; 1:200) and a rat anti-mouse Ly6G antibody (BioXCell, BE0075-1; 1:200) for 1 hour at room temperature. After washing, the sections were incubated with Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, A-11055; 1:200), Alexa Fluor 488 or Alexa Fluor 594 donkey anti-rat IgG (Invitrogen, A-21208 or A-21209; 1:200), and DAPI (Biolegend; 1:1000) for 1 hour at RT in the dark. Sections were then washed and mounted with Aqua-Poly/Mount (Polysciences). A Nikon Eclipse Ti-U fluorescent microscope was used to acquire images at a 64-bit data depth.
Zinc formalin fixative
Manufactured by Merck Group
Zinc Formalin Fixative is a laboratory reagent used for the preservation and fixation of biological samples. It contains a combination of zinc salts and formalin, which work together to stabilize and preserve the structural integrity of tissues and cells. The fixative is designed to be used in a variety of laboratory applications, such as histology, pathology, and microscopy, where the preservation of sample morphology is critical for analysis and diagnosis.
Automatically generated - may contain errors
Lab products found in correlation
Cd138 bv421, by BioLegend (2 mentions)
B220 af488, by Thermo Fisher Scientific (2 mentions)
Cd4 af594, by BioLegend (2 mentions)
Gl7 af647, by BD (2 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Eclipse ti u fluorescent microscope, by Nikon (1 mentions)
Ab6673, by Abcam (1 mentions)
Be0075 1, by BioXCell (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Xylene, by Merck Group (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Cd3 clone cd3 12, by Abcam (1 mentions)
Nanozoomer 2.0 rs digital slide scanner, by Hamamatsu Photonics (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Ivis spectrum, by PerkinElmer (1 mentions)
6 protocols using zinc formalin fixative
1
Immunofluorescence Analysis of Tumor Sections
2
Histological analysis of organ tissues
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Kidney, liver, and lung samples were fixed overnight with zinc formalin fixative (Sigma-Aldrich) and then embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin and eosin, Picro-Sirius red, or Masson’s trichrome (Microm-Microtech; France). Slides were scanned using a NanoZoomer 2.0-RS digital slide scanner and 2×, 10×, or 30× objective lenses with a numerical aperture of 0.75 (Hamamatsu Photonics), and images were analyzed using NDP View2 software (Hamamatsu Photonics). Immunohistofluorescence staining was performed on paraffin sections using primary antibodies against the pan-T cell marker CD3 (clone CD3-12, Abcam; 1/250) and the macrophage marker CD68 (PA5-89134, Thermo Fischer Scientific; 1/200), followed by staining with appropriate secondary antibodies Alexa Fluor™ 488 and Alexa Fluor™ 594 (Invitrogen, Thermo Fisher Scientific, 1/100). Nuclei were stained with Hoechst 33342 (Invitrogen; Molecular Probes; 1/300).
3
Spleen Tissue Immunostaining and Imaging Protocol
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After harvest, spleens were passed through a sucrose gradient (10% for 1
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).
4
Immunohistochemical Analysis of Xenograft Tumors
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Xenograft and liver sections were fixed overnight at 4 °C in zinc formalin fixative (Sigma, #Z2902). Five micrometre paraffin sections were stained with Ki67 (1:100), cleaved caspase 3 (1:100) and MT-CO2 (1:200), or Picro Sirius Red according to the suppliers’ recommendations. Fluorescently stained tissues from 6 tumors (for each genotype) were viewed by a Zeiss LSM 880 microscope (Zeiss, Thornwood, NY). Images (at least 5 images for each tumor) were quantified and analyzed using the Zen 2012 software (Zeiss, Thornwood, NY). Nuclei count per field was determined using the Zen 2012 software (6 images per animal, 5 animals per genotype).
Hematoxylin and Eosin (H&E) staining for routine histology was performed by Histoserv, Inc. (Gaithersburg).
Hematoxylin and Eosin (H&E) staining for routine histology was performed by Histoserv, Inc. (Gaithersburg).
5
Spleen Tissue Immunostaining and Imaging Protocol
6
Tumor Inoculation and Metastasis Quantification
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Tumor cells were harvested and single cell suspensions in 100 μL PBS were injected s.c. into right hind flank (for AB1 model) or into the second mammary gland (for 4T1 model) of BALB/c mice. Tumor volumes were measured by caliper (tumor volume = 1/2(length × width2)). Luciferase-expressing tumors were measured with IVIS spectrum (PerkinElmer) and presented as number of photons per second per square centimeter per steradian (photons/s/cm2/sr) within regions of interest (ROI) using Living Image software (version 4.0, PerkinElmer), as previously described.11 (link),32 (link) In the AB1 experimental metastasis model, 1 × 106 AB1 cells were injected into the tail vein of BALB/c mice, and the colonization of AB1 cells in the lung were determined by noninvasive bioluminescence imaging and H&E staining at the endpoint. AB1-Luc rechallenge was performed 60 days after primary tumor ablation on the opposite flank of animals. In the 4T1 spontaneous metastasis model, metastasis of 4T1 tumor cells into the lung are examined with a standard colonogenic assay at the endpoint.45 (link) Specimens were fixed in zinc formalin fixative (Sigma) and then embedded in paraffin blocks for H&E staining. Metastatic area was defined as the percentage of lung area occupied by metastatic tumor, measured by ImageJ.
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