Mast cells were stained with toluidine blue or with alcian blue and safranin. Briefly, PCLS were fixed with formaldehyde 4% (Roti−Histofix 4%, Carl Roth GMBH & Co. KG, Karlsruhe, Germany), dehydrated and embedded in paraffin. Then, 5 µm thick sections were cut, deparaffinized, rehydrated and incubated in 1% toluidin blue for 30 min. After rinsing with water, sections were dehydrated and coverslipped. Alternatively after rehydration, sections were incubated in 0.05% Alcian Blue in 0.02 M acetate buffer, pH 5.8 with 0.2 M MgCl for 4 h. After rinsing in water, sections were incubated for 5 min in 0.25% safranin 0.02 M acetate buffer, pH 5.0. After a final rinsing step, sections were dehydrated and coverslipped.
Roti histofix 4
Manufactured by Carl Roth
Sourced in Germany
Roti®-Histofix 4% is a ready-to-use fixative solution for the preservation and fixation of tissue samples. It is designed to maintain the structural integrity of the samples during the histological preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Amira 6, by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Carl Roth (3 mentions)
Triton x 100, by Merck Group (3 mentions)
μct50, by Scanco (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Sodium azide, by Merck Group (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
2 propanol, by Carl Roth (2 mentions)
Odyssey blocking buffer tbs, by LI COR (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Hematoxylin eosin staining, by Merck Group (2 mentions)
Anti epcam, by OriGene (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
78 protocols using roti histofix 4
1
Mast Cell Histological Staining
2
Fluorescence Imaging of Engineered Bone Tissue
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For imaging of fluorescence-labeled samples, scaffolds were collected on day 21 after transfer to osteogenic medium. The samples were washed with PBS and fixed with Roti®-Histofix 4% (#P0874, Carl Roth) for 10 min at RT. After this treatment, the scaffolds were permeabilized with 0.1% Triton X-100 (in PBS) for 5 min, submerged in staining solution (1:1000 DAPI/1:40 PF546-phalloidin in PBS), and kept light-protected for 25 min at RT. Both DAPI (#PKCA70740043) and PF546-phalloidin (PromoFluor546, #PKPF546701) fluorescence dyes were from PromoCell (Heidelberg, Germany). The samples were rinsed three times with PBS for 5 min after each preparation step. Sample preparation for imaging of hydroxyapatite (HA) particle formation was similar to DAPI/PF546-phalloidin staining. After fixation, permeabilization, and washing steps, the samples were incubated in a staining solution with 1:100 OsteoImage dye (OsteoImageTM Mineralization assay, #PA1503, Lonza, Basel, Switzerland) for 30 min at RT. After this step, the samples were washed with PBS three times for 5 min.
3
Immunofluorescent Staining of Actin and Nuclei
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To stain the actin cytoskeleton, and nuclei, cells were washed twice with ice-cold PBS and fixed with paraformaldehyde Roti-Histofix 4% (Roth, Karlsruhe, Germany) for 15 min. Cells were washed twice with ice-cold PBS for 5 min and incubated with ice-cold 0.1% Triton X-/PBS (Roth, Karlsruhe, Germany) for 5 min. Cells were washed twice with PBS and stained with 0.46% Bisbenzimide H 33258 (Sigma-Aldrich, Steinheim, Germany), and 1% Phalloidin-Atto-565 (Sigma-Aldrich, Steinheim, Germany). Cells were incubated for 1 h at RT in the dark. Afterward, cells were washed twice with PBS for 5 min and kept in PBS at 4°C until imaging. Images were acquired on a Leica DMi8 microscope using the HC PL APO ×63/1.40 oil objective. Images were processed using the Leica LasX software.
4
Hard Palate Tissue Preparation
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Tissue samples were fixed using a 4% phosphate-buffered formaldehyde solution (Roti®-Histofix 4%, Carl Roth, Karlsruhe, Germany) for 24 h at room temperature and cut into six segments of approximately 0.5-mm thickness: two blocks each for the anterior, median and posterior parts of the hard palate. The bone blocks were marked with a surgical suture for the distinction of anterior, median and posterior parts. Specimens were soaked in 10% Tris-buffered ethylenediaminetetraacetic acid (Carl Roth, Karlsruhe, Germany) and placed in an ultrasonic decalcifying automat at 37 °C for 14 days to speed up the process. Dehydration and clearing of the specimens was performed in a series of increasing concentrations of ethanol followed by three xylene immersions (TP1020, Leica, Wetzlar, Germany). The tissue segments were embedded in paraffin wax using a Leica embedding station (EG 1150H, Leica). Segments were cooled and consecutive paraffin blocks were cut along the longitudinal axis of the hard palate into serial, 4-μm-thick sections using a semi-automated precision microtome (Cut 5062, SLEE, Mainz, Germany).
5
Quantifying ER-Mitochondria Interactions via PLA
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Voltage-dependent anion channel 1 (VDAC1) and inositol 1,4,5-trisphosphate receptor type1 (IP3R1) proximity were measured by in situ proximity ligation assay (PLA) (Duolink II Fluorescence, OLink, Upsalla, Sweden) to detect, visualize and quantify ER-mitochondria interactions (33 (link)). Briefly, macrophages were plated at 1x105 cells/well in μ-Slide 8-well chambers (80826, Ibidi), fixed with ROTI histofix 4% (Carl Roth, Karlsruhe, Germany), permeabilized with Triton-X 100 (0.05% in phosphate-buffered saline (PBS)), blocked and incubated overnight with antibodies against VDAC1 (ab14734, Abcam) and IP3R1 (07-1213, Millipore). After washing, samples were incubated with the respective PLA-probes for one hour (37°C), washed and ligated for 30min (37°C). After an additional washing, amplification with polymerase was allowed for 100 min (37°C). The nuclei were stained using 1µg/ml DAPI (Sigma-Aldrich). Images were acquired by confocal microscope (LSM 800, Carl Zeiss). Protein-protein interactions are represented as individual fluorescent dots. 10 fields of about 20 cells per condition for each experiment were acquired, and quantification of detected PLA spots per cell was then analyzed using Image J software.
6
Preparing Collagen-Embedded HT1376 Cells
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At a confluency of 90% to 100%, HT1376 cells were collected and pelleted at 400×g for 5 minutes. Cells were resuspended in 50-μL collagen matrix (Cellmatrix Type I-A, #637–00653, Fujifilm Wako Chemicals) and supplemented with 10x Ham's F-12 and reconstitution buffer at a 8:1:1 ratio. The collagen-cell preparations were incubated at 37°C for 30 minutes until solidification. The collagen-cell pellet was then transferred into a plastic cassette with a swamp and fixed in ROTIHistofix 4% (P087.5, Carl Roth) for 2 hours at room temperature. Afterwards the samples were washed twice with tap water for 30 minutes, then transferred to 70% Ethanol and afterwards embedded in FFPE according to standard protocols. Subsequently, IHC staining of NECTIN-4 was performed using the VENTANA BenchMark ULTRA autostainer (Ventana) as described above.
7
DAPI-Based Cell Staining Protocol
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For DAPI staining, samples were transferred into a new 24-well culture plate after d1, d4 and d7, washed with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde (Roti-Histofix 4%, Carl Roth GmbH Karlsruhe, Germany). Samples were washed with PBS and distilled H2O and stained with 1000 µl DAPI mixture (1 µg/ml) (Roche Molecular Systems, Pleasanton, CA, USA) per sample. After repeated washing with distilled H2O, they were stored with 200 µl PBS per sample at 4 °C.
8
Immunocytochemistry in Cultured Neurons
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For immunocytochemistry, cells were pre-fixed in 2% formaldehyde for 5 min and then fixed in 4% formaldehyde in phosphate buffer (ROTI® Histofix 4%, Carl Roth) for 15 min. After washing with PBS, cells were stored at 4 °C until further use.
To reduce background fluorescence, cells were quenched with 0.1 M NH4Cl for 10 min at RT. Unspecific binding of antibodies was blocked and cells were permeabilized with normal donkey serum (017-000-121, Jackson ImmunoResearch)/0.3% (v/v) triton (Triton® X-100, Sigma-Aldrich) in PBS 0.01 M for 2 h at RT. Afterward, cells were incubated overnight at 4 °C in PBS 0.01 M/2% bovine serum albumin (001-000-161, Jackson ImmunoResearch)/0.05% sodium azide (S002, Sigma-Aldrich)/0.1% triton with the following primary antibody: rabbit monoclonal anti-NeuN (ab177487, Abcam,1:200). For fluorescence labeling, the following fluorophore-conjugated secondary antibody was used: Alexa Fluor647-conjugated AffiniPure anti-rabbit IgG (H + L) (Jackson ImmunoResearch 1:200).
Images from fixed neurons were taken with a 10 × objective (UPLFLN10X2PH, Olympus) with an epifluorescence microscope (IX81, Olympus) connected to a CCD camera (XM10, Olympus) using the cellSens software (Olympus). Images were subsequently analyzed in Fiji.
To reduce background fluorescence, cells were quenched with 0.1 M NH4Cl for 10 min at RT. Unspecific binding of antibodies was blocked and cells were permeabilized with normal donkey serum (017-000-121, Jackson ImmunoResearch)/0.3% (v/v) triton (Triton® X-100, Sigma-Aldrich) in PBS 0.01 M for 2 h at RT. Afterward, cells were incubated overnight at 4 °C in PBS 0.01 M/2% bovine serum albumin (001-000-161, Jackson ImmunoResearch)/0.05% sodium azide (S002, Sigma-Aldrich)/0.1% triton with the following primary antibody: rabbit monoclonal anti-NeuN (ab177487, Abcam,1:200). For fluorescence labeling, the following fluorophore-conjugated secondary antibody was used: Alexa Fluor647-conjugated AffiniPure anti-rabbit IgG (H + L) (Jackson ImmunoResearch 1:200).
Images from fixed neurons were taken with a 10 × objective (UPLFLN10X2PH, Olympus) with an epifluorescence microscope (IX81, Olympus) connected to a CCD camera (XM10, Olympus) using the cellSens software (Olympus). Images were subsequently analyzed in Fiji.
9
Histopathological Analysis of Brain and GI Tract
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For histopathological analysis, the right hemisphere of the brain and the gastrointestinal tract were collected and fixed in ROTI®Histofix 4% (4% formaldehyde, Roth) for a minimum of 48 h. Two longitudinal sections of the brain and representative sections of duodenum, jejunum, ileum, caecum, colon and rectum of all mice were embedded in paraffin wax followed by cutting 2-3 µm thick sections using a microtome. Sections were stained with hematoxylin and eosin (H&E).
10
Characterization of Cellular Interactions with Carbon Nanotubes
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SWCNTs and MWCNTs were purchased from Carbon Solutions (Riverside, CA, USA). Acetic acid, chitosan, Sirius Red, picric acid, formaldehyde and tetramethylrhodamine (TRITC)–phalloidin were purchased from Sigma-Aldrich (St Louis, MO, USA). DMEM 1× solution with high glucose, DMEM powder with high glucose, FBS, penicillin/streptomycin and PBS were purchased from Gibco (Waltham, MA, USA). Sterile water (Aqua®) was purchased from Braun (Kronberg im Taunus, Germany). Acid-soluble collagen was purchased from Devro (Chryston, UK). Roti®-Block, Triton X-100 and Roti®-Histofix 4% were purchased from Carl Roth (Karlsruhe, Germany), DAPI was purchased from Hoffman-La Roche Ltd. (Basel, Switzerland). The 3T3 fibroblast cell lines were a kind gift from Dr Johnny Amer (American Type Culture Collection [ATCC], Manassas, VA, USA) and human foreskin fibroblasts were obtained from ATCC.
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