Invasion assays were performed as previously described to evaluate the invasiveness of cancer cells (34 (link)). Briefly, we coated the upper membranes of cell culture inserts (Cat# 3401, Corning Incorporated) with Matrigel (Cat# A14132-01, Gibco) for 1 h. Serum-free regular medium (described in cell culture) of 200 µl was plated to the upper compartment, and 500 µl regular medium containing 10% FBS was added to the lower compartment. The cells were plated at a density of 2×104 cells/ml in the upper inserts and incubated at 37°C for 24 or 48 h. The upper membranes containing invading cells were fixed using 100% methanol for 20 min and stained for 15 min with 0.1% crystal violet (Sigma Chemicals) at room temperature. The upper surface of the inserts was washed in PBS, and noninvasive cells were wiped with cotton swabs. The membranes containing invading cells were mounted on slides, and light microscopy (100×, magnification, Olympus BX51-p polarizing Microscope) was used to count the number of cells present.
Bx51p polarizing microscope
Manufactured by Olympus
Sourced in United States
The BX51P is a polarizing microscope designed for a range of applications. It features an integrated polarizer and analyzer for examining birefringent samples. The microscope provides high-quality optical performance and durability for routine laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (1 mentions)
Cell culture insert, by Corning (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Saponin, by Nacalai Tesque (1 mentions)
Goat serum, by Fujifilm (1 mentions)
Zinc formalin, by Polysciences (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Anti mucin2, by Santa Cruz Biotechnology (1 mentions)
Eosin solution, by Fujifilm (1 mentions)
Leica tcs sp8, by Leica Microsystems (1 mentions)
5 protocols using bx51p polarizing microscope
1
Invasion Assay for Cancer Cell Invasiveness
2
Histological and Immunohistochemical Analysis of Distal Colon
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For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).
3
Quantitative Analysis of Collagen
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The quantitative analysis of collagen was performed using an BX51P polarizing microscope (Olympus, New York, NY, USA) with a plan achromatic objective, coupled to a camera (Sony CCD-Iris, model DXC-107ª, Tokyo, Japan) and a PC, including a Pentium 233MMX processor and a plate scanner. Images were analyzed in the software CorelDraw Graphics Suite X4 (version 12, Ottawa, ON, Canada) and measured interactively with the original image using the software Image Tool (version 3.00, developed University of Texas Health Science Center, San Antonio, TX, USA (UTHSCSA)) for Windows, available freely online from the University of Texas Health Science Center at San Antonio. The image was evaluated in greatest staining (“hot spot”) in the tissue sections, at ×400 magnification. Collagen staining was quantified as the percentage ratio of the area of staining in the studied image, as compared to the total area of the digitized field. For qualitative assessment, collagen was scored by a blinded examiner as mature collagen (well organized and thick collagen fibers) or immature collagen (loosely dispersed and poorly organized thin collagen fibers).
4
Histopathological Evaluation of Vaginal Tissue
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The Histopathology study was done only for combination form of drugs either in soluble form or nanoformulation (ECNPs). Doses of soluble drugs in combination and ECNPs were given as described in dose schedule (n=3). Negative and positive controls animals were treated with saline and 10 mg/kg nonoxynol-9 (N-9) respectively. Multiple doses were repeated up to three doses at time gap of 2 h. After the completion of measurements at the time points, animals were sacrificed under standard protocol. Vaginal tissue was fixed in 10% Neutral Buffered Formalin, followed by sectioning using cryomicrotome and Hematoxylin& Eosin staining. All pictures included in the results section were taken at 100× zoom using Olympus BX51P polarizing microscope.
5
Arthrocentesis and Crystal Analysis
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Arthrocentesis and synovial fluid analysis were performed by one of two experienced rheumatologists (VSS or PK) using an Olympus BX51-P polarizing microscope from 2016. CPLM was performed directly after arthrocentesis. The presence of negatively birefringent needle-shaped crystals was classified as gout while positively birefringent rhomboid-shaped crystals were classified as CPPD. Joint aspirate was analysed visually and sent for microbiological analysis to rule out septic arthritis.
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