Tissues were fixed overnight in 10% formalin, de-paraffinised in xylene and then rehydrated in 70% ethanol. Antigen retrieval was performed in citrate buffer, pH 6.0, or Tris-EDTA, pH 9.0, for 30 min, and then blocked for 1 hour in 10% donkey serum diluted in Tris-buffered saline 0.1% Triton with 0.025% Tween-20 (TBST-TW). For immunofluorescence (IF), the primary antibodies were incubated in 1% bovine serum albumin (BSA) TBS-TW overnight at 4°C. Secondary antibodies (1:500) were incubated for 1 hour in 1% BSA with 0.025% Tween-20 (TBS-TW) at room temperature (RT) in the dark. For IHC, primary antibodies were incubated in 1% BSA Tris-buffered saline 0.1% Tween (TBS-T) overnight at 4°C. Slides were incubated in horseradish peroxidase (Cell Signaling, Danvers, Massachusetts) secondary antibody (1:300) for 60 min in 1% BSA TBS-T for 1 hour at RT. Slides were then incubated with diaminobenzidine (Vector, Burlingame, California) followed by counterstaining with haematoxylin (Leica, Buffalo Grove, Illinois). Dilutions of primary antibodies are listed in online supplemental table 2 . Slides were mounted with ProLong Gold Antifade Mounting Medium with 4’, 6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were photographed on an Olympus BX53F microscope with an Olympus digital camera (Center Valley, Pennsylvania).
Haematoxylin
Manufactured by Leica
Sourced in Germany
Haematoxylin is a natural dye derived from the heartwood of the Logwood tree. It is a commonly used stain in histology and cytology laboratories for the visualization of cellular structures. Haematoxylin stains cell nuclei a distinctive blue-purple color, allowing for the identification and analysis of various cell types and tissue structures.
Automatically generated - may contain errors
Lab products found in correlation
Blue buffer, by Leica (3 mentions)
Rm2135 microtome, by Leica (2 mentions)
Axio imager a2, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Histogel, by Thermo Fisher Scientific (2 mentions)
Zen software, by Zeiss (2 mentions)
Eosin y 515, by Leica (2 mentions)
Tissuetek vip tissue processing system, by Sakura Finetek (2 mentions)
Selectech staining solutions, by Leica (2 mentions)
Digital camera, by Olympus (1 mentions)
Bx53f microscope, by Olympus (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Prolong gold antifade mounting medium with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Nanozoomer 2.0 ht scan system, by Hamamatsu Photonics (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Dm6000 microscope, by Leica (1 mentions)
Novared revelation kit, by Vector Laboratories (1 mentions)
Immpress anti rat ig, by Vector Laboratories (1 mentions)
Microtome, by Leica (1 mentions)
12 protocols using haematoxylin
1
Immunohistochemistry and Immunofluorescence Protocols
2
Immunohistochemical Tissue Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue staining was performed on formalin fixed, paraffin embedded 3 µm-thick tissue slices according to standard procedures. Following dewaxing, rehydrating, exposure to antigen retrieval buffer (Dako, Agilent, Santa Clara, CA, USA) and blocking of endogenous peroxidases (Dako REAL®, Agilent), primary antibodies in a background-reducing diluent (Agilent) were applied at 4°C overnight. The diluent with no antibody was used as a negative control. Appropriate biotinylated secondary antibodies were applied at room temperature for 30 minutes, followed by the avidin-biotin complex (Vector Labs, Burlingame, CA, USA) and 3′,3′-diaminobenzidine (DAB+, Agilent). Cell nuclei were counterstained with haematoxylin (Leica, Wetzlar, Germany). All antibodies are listed in Table 2
Histological images were captured at 20× or 40× magnification (resolution 0.46 μm/pixel) using the Hamamatsu NanoZoomer 2.0-HT Scan System (Hamamatsu Photonics, Hamamatsu, Japan). All staining was digitally analysed using Aperio® Precision Image Analysis Software and Image Scope version 11 (Aperio® Technologies, Inc., Vista, CA, USA), applying algorithms, as described previously (47 (link)).
Histological images were captured at 20× or 40× magnification (resolution 0.46 μm/pixel) using the Hamamatsu NanoZoomer 2.0-HT Scan System (Hamamatsu Photonics, Hamamatsu, Japan). All staining was digitally analysed using Aperio® Precision Image Analysis Software and Image Scope version 11 (Aperio® Technologies, Inc., Vista, CA, USA), applying algorithms, as described previously (47 (link)).
3
F4/80+ Macrophage Quantification in eWAT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded eWAT tissues were serially sectioned into 5 µm slices. Immunohistochemistry (IHC) was performed after deparaffinization, rehydration, and antigen retrieval by heating in 10 mM sodium citrate buffer (pH 6, 95 °C). Sections were then saturated in a 3% BSA solution containing 3% hydrogen peroxide to block endogenous peroxidase activity. After saturation, sections were incubated with a rat anti-mouse F4/80 antibody (1/100; AbD Serotec, MCA497) for 1 h at room temperature. Sections were further incubated with ImmPress anti-rat Ig (Vector Laboratories, MP7444). IHC staining in sections was visualized using a NovaRed revelation kit (Vector Lab, SK4800) and a Leica DM 6000 microscope (Leica Microsystems). Negative controls were performed by incubating sections without the primary antibody. All sections were counterstained with haematoxylin (Leica, 380156E). Positive F4/80 infiltrates were counted in 5 different fields per section and two different sections per group. Adipocyte size distribution frequencies and mean sizes were evaluated in stained sections.
4
Histopathological Analysis of Chicken Brain and Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sampled chicken brains and livers were fixed in 10% buffered formaldehyde, pH 7.2 (Sigma-Aldrich), then dehydrated in a graded series of ethanols, embedded in paraffin (Paraplast, Sigma-Aldrich), cut into sections (5 μm) with a microtome (Leica, Nussloch, Germany) and stained using haematoxylin (POCH S.A, Gliwice, Poland) and eosin (BDH Laboratory Supplies, Poole, Dorset, United Kingdom). Morphology was evaluated using a light microscope (DM750; Leica) connected to a digital camera and a computer analysis system (Leica).
5
Immunohistochemical Staining of VDR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After preparation of the slides, antigen retrieval was achieved by incubating the slides for 15 min in hot preheated sodium citrate (pH 6.0) buffer and 30 min of cooling at room temperature. Endogenous peroxidases were quenched by incubating the slides in 3% hydrogen peroxide for 10 min, followed by three rinses with HBSS, and incubation for 1 hour in 3% BSA + 1% goat serum in HBSS to reduce nonspecific background. Primary antibodies VDR (1:300) were applied for overnight in a cold room. The secondary antibody (1:100, Jackson ImmunoResearch Laboratories, Cat.No.115-065-174, West Grove, PA, USA). The slides were incubated with vectastain ABC reagent (Vector Laboratories, Cat.No. PK-6100, Burlingame, CA 94010, USA) for 1 hour. After washing with HBSS, color development was achieved by applying a peroxidase substrate kit (Vector Laboratories, Cat.No. SK-4800, Burlingame, CA, USA). The duration of peroxidase substrate incubation was determined through pilot experiments and was then held constant for all of the slides. After washing in distilled water, the sections were counterstained with haematoxylin (Leica, Cat.No.3801570, Wetzlar, Germany), dehydrated through ethanol and xylene, and cover‐slipped using a permount (Fisher Scientific, Cat.No. SP15-100, Waltham, MA, USA).
6
Histochemical Analysis of Liver and Intestine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen liver sections (10 µm) were stained with Oil red O (Sigma-Aldrich), as described previously (17) . Liver sections (5 µm) embedded in paraffin were stained with haematoxylineosin (Sigma-Aldrich) to evaluate the histologic features as detailed before (14) . To determine goblet cell number being indicative of mucous formation (18) , duodenal sections (4 µm) embedded in paraffin were consecutively stained with Alcian blue (30 min), periodic acid (10 min), Schiff's reagent (15 min) (all from Carl-Roth) and counterstained with haematoxylin. Quantitative evaluation of staining was carried out as previously described in detail (19) . Representative photomicrographs of Oil red O (100×) and haematoxylin-eosin (200×), as well as goblet cell (200×) staining, were captured using the system incorporated in the microscope (Leica DM4000 B LED).
7
Immunohistochemical Assessment of ABCB1 in Medulloblastoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) was performed using the Dako Envision Detection kit (DAKO REAL EnVision) [53 (link)]. Sections were counter-stained with haematoxylin (Leica Microsystems). As negative controls, adjacent or similar sections were processed with antibody diluent (Dako). The antibodies (Abs) used to stain the original patient samples with appropriate control for each Ab are summarised in Additional file 2 : Table S2.
A total of 27 Nottingham MB patients diagnosed between January 1986 and January 2006 were analysed by IHC. Consent for tumour tissue use was taken in accordance with national tumour banking procedures (UK: 05/MRE/04/70). A second TMA comprising 233 patient samples was obtained from the DKFZ [48 (link)]. Both TMAs were stained with the anti-ABCB1 Ab (C219, 1:40; Calbiochem) and patients with clear evidence of cell membrane staining were scored as positive by three independent reviewers who were blinded to clinical and patient molecular variables.
A total of 27 Nottingham MB patients diagnosed between January 1986 and January 2006 were analysed by IHC. Consent for tumour tissue use was taken in accordance with national tumour banking procedures (UK: 05/MRE/04/70). A second TMA comprising 233 patient samples was obtained from the DKFZ [48 (link)]. Both TMAs were stained with the anti-ABCB1 Ab (C219, 1:40; Calbiochem) and patients with clear evidence of cell membrane staining were scored as positive by three independent reviewers who were blinded to clinical and patient molecular variables.
8
Optimizing RNA Preservation for LCM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sections of 8 µm thickness were cut using a cryostat (Leica Microsystems, Milton Keynes, UK) at -25°C and adhered onto MembraneSlides (Molecular Machines & Industries AG, Glattbrugg, Switzerland) pre-cooled inside the cryostat and maintained dehydrated in -80°C freezer until needed. All slides, LCM caps, and solutions were obtained from Molecular Machines & Industries. The slides were dehydrated in graded alcohols and xylene and placed in a desiccator until laser capture. The sections were stained with (a) Acridine orange (Life Technologies), (b) Alcoholic cresyl violet (Life Technologies), (c) Alcoholic cresyl violet and alcoholic eosin Y (Sigma-Aldrich), (d) Arcturus Paradise stain (Life Technologies) in combination with RNAse inhibitor, (e) Arcturus Paradise stain, (f) haematoxylin (Leica Microsystems, Milton Keynes, UK) and alcoholic eosin Y in combination with RNAse inhibitor, and (g) haematoxylin and alcoholic eosin Y together with alcoholic eosin-Y. RIN values were assessed using Nano/Pico Chips on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) (Clement-Ziza et al. 2008) . Having identified the optimal stain, LCM was performed in 0, 30 and 50 min to assess the effect of duration of LCM on RIN values.
9
Kidney Organoid Histological Processing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney organoids were fixed overnight at 4°C in 2% or 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), pre-embedded in HistoGel (Thermo Fisher, Carlsbad, CA), then dehydrated and infiltrated with paraffin using a TissueTek VIP tissue processing system (Sakura Finetek USA, Torrance, CA). Planar or transverse 5μm sections were obtained using a Leica RM 2135 microtome (Leica Biosystems, Buffalo Grove, IL). Sections were baked, de-paraffinized and hydrated to water prior to staining following a standard regressive staining protocol using SelecTech staining solutions (Leica Biosystems, Richmond, IL; Haematoxylin #3801570, Define #3803590, Blue Buffer #3802915, and Eosin Y 515 #3801615). Stained slides were serially dehydrated, cleared, and mounted in Permaslip (Alban Scientific Inc, St. Louis, MO #6530B). Images were acquired on a Zeiss Axio Imager A2 with Zeiss Zen software (Zeiss Microscopy, Thornwood, NY).
10
Immunohistochemical Analysis of Ki-67 in Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining was performed with monoclonal mouse anti-human Ki-67 antibody (clone MIB-1, M7240; dilution 1:100; Agilent Technologies) in a Leica BOND-III IHC autostainer, with antigen retrieval performed at pH 9.0 by using a Leica Epitope retrieval solution 2 for 20 min at 100°C. Slides were counterstained with haematoxylin (Leica Biosystems) and mounted with ClearVue mounting medium (Thermo Fisher Scientific).
Ki-67 analysis was evaluated using manual analysis (MA) of 'hot spots' (Yamazaki et al. 2016 (link)). Hot spots, which contained the most frequent Ki-67-positive cells, were selected through scanning the whole immunostained slide preparation. MA was performed using the ×40 microscope objective of 1000 cells in hot spot areas. The Ventana Benchmark mismatch repair panel (MSH2 (G219-1129) and CONFIRM anti-MSH6) was performed on 4-µm sections of paraffin-embedded tumour tissue in accordance with the manufacturer’s guidelines and interpreted by an experienced pathologist (AM).
Ki-67 analysis was evaluated using manual analysis (MA) of 'hot spots' (Yamazaki et al. 2016 (link)). Hot spots, which contained the most frequent Ki-67-positive cells, were selected through scanning the whole immunostained slide preparation. MA was performed using the ×40 microscope objective of 1000 cells in hot spot areas. The Ventana Benchmark mismatch repair panel (MSH2 (G219-1129) and CONFIRM anti-MSH6) was performed on 4-µm sections of paraffin-embedded tumour tissue in accordance with the manufacturer’s guidelines and interpreted by an experienced pathologist (AM).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!