Totally, 5 μm FFPE sections were dewaxed, treated with H2O2 before performing antigen retrieval. Sections were boiled in citrate buffer (Sigma; C9999) for 30 min. Sections were washed in PBS Tween, then blocked 50% FBS for 1 h. After washing, sections were probed with anti CD68 (1:100) and anti CXCL5 (1:50) for 1 h. After further washing, sections were stained with donkey anti-mouse 488 (1:1000) and donkey anti goat 557 (1:200) in 10% FBS and incubated for 1 h. Final washes were performed, and sections were stained with DAPI for 30 s before being mounted (Vector Labs; H-100). Sections were scanned using an Olympus BX61VS and images were analysed using OylVIA software.
H 100
Manufactured by Vector Laboratories
The H-100 is a compact and versatile lab equipment designed for general laboratory use. It features a durable stainless-steel construction and a digital control panel for precise temperature regulation. The H-100 is capable of maintaining a consistent temperature range to support various laboratory processes.
Automatically generated - may contain errors
4 protocols using h 100
1
Immunohistochemical Staining Protocol for CD68 and CXCL5
2
Larvae Immunostaining and Confocal Imaging
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Larvae were dissected in 1X PBS, followed by fixing in 4% paraformaldehyde (Himedia—TCL-119 - 100ml) for 30 minutes at room temperature and three washes in 1X PBS with 0.2% TritonX-100 (Himedia—MB031, 1X PBST). Primary antibodies were incubated overnight at 4°C. Followed by blocking in 5% normal goat serum (Himedia—RM10701) for 1h at room temperature and then secondary antibody incubation followed by washing and dissection. Samples were mounted in Vectashield (VectorLabs—H100) and imaged under 40X or 63X oil immersion Leica Stellaris 5 or Olympus FV3000 confocal microscopes. Images were processed using Fiji. All antibody dilutions and the blocking solution were made in 1X PBST; details of antibodies and their dilutions used are listed in Table 2 .
3
Tumor Vasculature Characterization by Immunofluorescence
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Tumor tissue samples were cut into thin pieces and digested with 20 mm proteinase K for 5 min, followed by incubation for 30 min with 100% methanol. Tissue samples were further incubated at 4 °C overnight with 0.3% Triton X‐100 PBS containing 3% skim milk. Samples were rigorously washed with PBS and subsequently incubated overnight with a combination of a rat anti‐mouse CD31 (1:200, Cat. 553 370, BD Pharmingen) antibody and a rabbit polyclonal anti‐NG2 (1:200, Cat. AB5320, Merck) antibody. After rigorous washes with PBS, species‐matched secondary antibodies were added for incubation overnight at 4 °C. After washing with PBS, stained tissue samples were mounted by a vectashield‐mounting medium (H100, Vector Laboratories). Images were taken by confocal microscopy (Nikon, DS‐QilMC). 3D images of tumor vessels were analyzed. Positive signals of CD31 and NG2 were calculated by Adobe Photoshop software.
4
Tribolium Brain Dissection and Processing
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Tribolium embryos were collected, fixed and stored as described (Buescher et al., 2020) (link).
Tribolium larval, pupal and adult brains were dissected in ice-cold 1x phosphate-buffered saline (PBS) for up to 30 min. Then methanol-free formaldehyde was added to a final concentration of 4% (v/v). Fixation was performed for 30 (larval-), 45 (pupal-) or 60 (adultbrains) min on ice. Subsequently, the brains were washed 3x for 20 min each with ice-cold 1x
PBST (PBS including 0.1% Triton-X100). In the second wash, DAPI was added to a final concentration of 1ng/l. Brains not dedicated to immunohistochemistry were mounted in VectaShield H-100 and imaged immediately. Brains dedicated to immunohistochemistry were placed into blocking solution containing 3% (w/v) bovine serum albumin (BSA) and 0.05% sodium azide. Adult brains dedicated to RNA in situ hybridization were dehydrated by putting them through an ethanol series: 25% ETOH:75% PBS, 50% ETOH:50% PBS, 75%
ETOH:25% PBS for 5 min incubation each. Finally, the brains were placed in 100% ETOH and kept at -20 o C for several days prior to in situ hybridization.
Tribolium larval, pupal and adult brains were dissected in ice-cold 1x phosphate-buffered saline (PBS) for up to 30 min. Then methanol-free formaldehyde was added to a final concentration of 4% (v/v). Fixation was performed for 30 (larval-), 45 (pupal-) or 60 (adultbrains) min on ice. Subsequently, the brains were washed 3x for 20 min each with ice-cold 1x
PBST (PBS including 0.1% Triton-X100). In the second wash, DAPI was added to a final concentration of 1ng/l. Brains not dedicated to immunohistochemistry were mounted in VectaShield H-100 and imaged immediately. Brains dedicated to immunohistochemistry were placed into blocking solution containing 3% (w/v) bovine serum albumin (BSA) and 0.05% sodium azide. Adult brains dedicated to RNA in situ hybridization were dehydrated by putting them through an ethanol series: 25% ETOH:75% PBS, 50% ETOH:50% PBS, 75%
ETOH:25% PBS for 5 min incubation each. Finally, the brains were placed in 100% ETOH and kept at -20 o C for several days prior to in situ hybridization.
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