Samples were fixed in 2.5% paraformaldehyde in 0.025 mM phosphate-buffered saline (PBS) and evacuated using a vacuum pump for 12 h. Fixed samples were dehydrated through the following series of EtOH concentrations: 30, 50, 70, 80, and 90% for 20 min each, and then 95 and 100% twice for 30 min. EtOH in dehydrated samples was exchanged for Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany) through the following series of Technovit 7100:EtOH: 1:4, 2:3, 3:2, and 4:1 each for 30 min, and then 100% Technovit for 30 min and 12 h. Samples were then solidified in Technovit 7100 resin following the manufacturer’s protocol. Embedded samples were cut into 5 μm sections using a microtome and a glass knife.
Technovit 7100 resin
Manufactured by Kulzer
Sourced in Germany
Technovit 7100 is a cold-curing, two-component, methacrylate-based resin for the embedding of samples in light microscopy. It is designed for the preparation of undecalcified tissue samples and other solid specimens.
Automatically generated - may contain errors
Lab products found in correlation
Microm hm 355, by Thermo Fisher Scientific (4 mentions)
Ds vi1 camera, by Nikon (4 mentions)
Jsm 6380, by JEOL (3 mentions)
Eclipse ci microscope, by Nikon (3 mentions)
Bx51 microscope, by Olympus (3 mentions)
Toluidine blue o, by Merck Group (3 mentions)
Em uc7, by Leica (3 mentions)
Reichert em ultraincised, by Leica (2 mentions)
Cacodylate buffer, by Polysciences (2 mentions)
Axio scope a1, by Zeiss (2 mentions)
Formaldehyde, by Polysciences (2 mentions)
Glutaraldehyde, by Polysciences (2 mentions)
Rm1215rt, by Leica (2 mentions)
Rm2235, by Leica (2 mentions)
Eclipse 50i, by Nikon (2 mentions)
Ds 5mc l2, by Nikon (2 mentions)
Rm2135, by Leica (2 mentions)
Axioimager microscope, by Zeiss (2 mentions)
Axiocam mrm camera, by Zeiss (2 mentions)
Axioplan 2 microscope, by Zeiss (2 mentions)
79 protocols using technovit 7100 resin
1
Technovit Embedding and Sectioning Protocol
2
Leaf Anatomy Ultrastructural Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis, the second leaf was taken from the apical bud of the shoot. Then, 5.0 × 5.0 mm sections were cut out from the middle part of the middle lobe of the trifoliate leaf, avoiding the midrib. The collected leaf materials were fixed in FAA (formalin: glacial acetic acid: 95% ethanol, 7:7:100 (v/v/v)) before storage in 70% ethanol. After that, the leaves were dehydrated using a graded ethanol series. Dehydrated leaf materials were sequentially passed through mixtures made of 95% ethanol/Technovit 7100 resin (Heraeus Kulzer, Germany) combinations (7:3, 15 h; 1:1, 15 h; 3:7, 15 h (v/v)) and then 100% Technovit for 15 h. The samples were then solidified in Technovit 7100 resin following the manufacturer’s protocol. Embedded samples were cut on an HM-325 rotary microtome (Microm, Germany) into sections of 7 μm thickness, placed onto slide glass, and dried on an electric slide warmer for 12 h. Dried slides were stained with 0.2% Sudan black B (Servicebio Technology Co., Ltd., Wuhan, China) for 60–90 s, rinsed with running water, and again dried by means of the electric slide warmer for more than 6 h to remove water. The stained slides were then mounted with Mowiol® (Sigma-Aldrich, Taufkirchen, Germany) and examined under a VHX-5000 digital light microscope (Keyence, Osaka, Japan). For each type of sample, 300 morphometric measurements were carried out.
3
Detailed Pollen-Pistil Interaction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The number of pollen grains that adhered to pistils was counted as described by Zinkl et al. (1999) , except that self-pollinated pistils were washed with 200 mL of 50 mM potassium phosphate, pH 7.4, before staining. To observe semithin sections of anthers, bud clusters were fixed with 2% (w/v) glutaraldehyde in 20 mM sodium cacodylate, pH 7.4, overnight at 4°C. The samples were dehydrated through an ethanol series and embedded in Technovit 7100 resin (Kulzer) according to the manufacturer's instructions. Sections were cut to a thickness of 2 mm using a Leica RM2255 microtome (Leica Biosystems), stained with 0.5% (w/v) Toluidine Blue O in 0.1% (w/v) Na 2 CO 3 , and observed with a BX51 microscope (Olympus) with bright-field illumination. Each sample was analyzed at least in two biological and two technical repeats with similar results.
Histochemical GUS staining was performed as described by Preuss et al. (1994) . To prepare sections, GUS-stained samples were embedded in Technovit 7100 resin (Kulzer). Each sample was analyzed at least in two biological and two technical repeats with similar results.
Histochemical GUS staining was performed as described by Preuss et al. (1994) . To prepare sections, GUS-stained samples were embedded in Technovit 7100 resin (Kulzer). Each sample was analyzed at least in two biological and two technical repeats with similar results.
4
GUS Staining of Plant Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Incised in orescence stem samples were soaked in ice-cold 90% (v/v) acetone for 15 min and then were washed three times with GUS buffer (0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM K 4 [Fe(CN) 6 ], 20% (v/v) methanol in 50 mM phosphate buffer, pH 7.2). After washing, the samples were incubated with GUS staining buffer (0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM K 4 [Fe(CN) 6 ], 20% (v/v) methanol, X-Gluc in 50 mM phosphate buffer, pH 7.2) at 37°C overnight. Samples were washed three times with 70% (v/v) ethanol, stained, and observed using a digital microscope (VH-S30B; Keyence). GUS-stained samples were dehydrated in 90% and 100% ethanol and embedded in Technovit 7100 resin (Heraeus Kulzer; http://heraeus-kulzer.com/en/int/home_4/worldmap_1.aspx). Tissue sections (20-30 µm thick) were prepared using an ultramicrotome with a glass knife (Reichert EM-Ultraincised; Leica; http://www.leica.com). The tissue reunion region was observed under a light microscope (DMRB; Leica).
5
Tissue Fixation and Sectioning for Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Incised in orescence stem samples were immersed overnight in a xative solution consisting of 2.5% (v/v) glutaraldehyde and 10% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 6.8-7.4). After xation, samples were washed four times in 0.1 M phosphate buffer, dehydrated in 30%, 50%, 70%, 90%, and 100% ethanol, and embedded in Technovit 7100 Resin (Heraeus Kulzer; http://heraeus-kulzer.com/en/int/home_4/worldmap_1.aspx). Tissue sections (3.0 µm thick) were prepared using an ultramicrotome with a glass knife (Reichert EM-Ultraincised; Leica; http://www.leica.com/). The sections were stained with 0.1% (w/v) toluidine blue O and the region of tissue reunion was observed under a light microscope (DMRB; Leica).
6
Ovule Morphology Characterization in F3 Plants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 0 DAA, two ovaries were sampled in each of the three F3 plants that were homozygous for the ‘MPK-1’ allele and the three F3 plants that were homozygous for the ‘Micro-Tom’ allele at the Pat-k locus. In all the F3 plants we used, the genotype at the nearest marker of two QTLs for number of seeds on chromosome 2 and 4 were ‘MPK-1’ homozygous.
Ovaries were fixed with formalin-acetic acid-alcohol: 50% ethanol, glacial acetic acid, and formalin at the ratio of 18:1:1 (v/v/v). After one night, the ovaries were washed with running tap water and then dehydrated with a series of ethanol treatments: 30%, 70%, 90%, 99%, and 100% ethanol (v/v). After substituting ethanol with Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany), the samples were embedded in resin and coagulated in a mold. Transverse sections (2 μm thick) were prepared with a rotary microtome and then stained with toluidine blue O to observe the morphology of ovules with light microscopy. Based on this observation, ovules were separated into the following two types: normal ovule (with micropyle that was properly closed) and abnormal ovule (with micropyle that had a cavity). We observed 10–12 ovules in each plant and evaluated ovule aberrancy as the percentage of the number of abnormal ovules to all observed ovules.
Ovaries were fixed with formalin-acetic acid-alcohol: 50% ethanol, glacial acetic acid, and formalin at the ratio of 18:1:1 (v/v/v). After one night, the ovaries were washed with running tap water and then dehydrated with a series of ethanol treatments: 30%, 70%, 90%, 99%, and 100% ethanol (v/v). After substituting ethanol with Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany), the samples were embedded in resin and coagulated in a mold. Transverse sections (2 μm thick) were prepared with a rotary microtome and then stained with toluidine blue O to observe the morphology of ovules with light microscopy. Based on this observation, ovules were separated into the following two types: normal ovule (with micropyle that was properly closed) and abnormal ovule (with micropyle that had a cavity). We observed 10–12 ovules in each plant and evaluated ovule aberrancy as the percentage of the number of abnormal ovules to all observed ovules.
7
Root and Leaf Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The harvested samples of fine roots and leaves were immediately fixed in a mix 2% (v/v) glutaraldehyde (pH 6.8; Polysciences, Warrington, USA) and 2% (v/v) formaldehyde (pH 6.8; Polysciences, Warrington, USA). After an overnight incubation in fixative solution, the samples were rinsed three times with a cacodylate buffer (0.05 M; pH 6.8; Polysciences) and then dehydrated in a graded ethanol series (10–100%, v/v). Subsequently, the samples were incubated in a series of ethanol:Technovit 7100 resin mixture (Heraeus Kulzer, Wehrheim, Germany) with ratios of 3:1, 1:1, 1:3, and finally in pure Technovit 7100 resin. Cross-sections were cut with a Leica RM2265 Fully Automated Rotary microtome (Leica-Reichert, Bensheim, Germany) at a thickness of 10 μm. The cross-sections were stained with 1% (m/v) aniline blue and examined under a light microscope (Axioscope A1, Carl Zeiss, Jena, Germany).
8
Anther Morphology Observation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anthers for morphological observation were collected from spikelets of the main panicles just before flowering. They were fixed in an FAA solution (formaldehyde: acetic acid: 70% ethanol = 1:1:18 (volume ratio)) with vacuum infiltration and were preserved at 4 °C. An ethanol series (70%, 80% and 90% ethanol) was used to dehydrate the samples for 2 days at each stage. Then, the samples were embedded in Technovit 7100 resin (Heraeus Kulzer, Germany), according to manufacturer’s instructions, and were cut into 3-µm sections with a rotary microtome, RM1215RT (Leica Biosystems, Germany). The sections were then stained with toluidine blue O solution, observed under a microscope, and photographed with a digital camera using the imaging software.
9
Histological Analysis of Spikelet Abscission Layer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The abscission layer (axial images of detached spikelet) was examined using a LEICA S6D microscope and photographs were taken with the MC170HD and Leica Application Suite (Leica, Germany). The samples for histological analysis were collected from the pedicel tissue of grains before heading, following Htun et al. ( 2014) and Inoue et al. (2015) (link). The samples were fixed in FAA solution (formaldehyde: acetic acid: 70% ethanol = 1:1 :18 (volume ratio)) with vacuum infiltration and were preserved at 4°C. They were dehydrated in an ethanol series (70%, 80%, and 90% ethanol) for 2 days at each stage and then embedded in Technovit 7100 resin (Heraeus Kulzer, Germany), according to the manufacturer's instructions. The samples were cut into 3-µm sections with a rotary microtome, RM1215RT (Leica Biosystems, Germany), and stained with toluidine blue O solution. These sections were observed under a microscope and photographed with a digital camera using the imaging software, ToupView (×86) (Amscope.com, US).
10
Histological Analysis of Plant Flowers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological observations were conducted for flowers collected in Hongo campus of the University of Tokyo (Tokyo, Japan) in 2022, by embedding samples in Technovit 7100 resin (Heraeus Kulzer, Hanau, Germany) after fixation with FAA [5% (v/v) acetic acid, 45% (v/v) EtOH, and 5% (v/v) formalin] overnight (or longer) and sequential dehydration with ethanol, as described in previous literature20 (link). Resin-embedded specimens were sliced by Leica Rotary Microtome (Leica microsystems, Wetzlar, Germany). 3–6 μm thick histological sections of were placed on cover slips, stained with 0.1% (w/v) toluidine blue in 0.1 M phosphate buffer (pH 7.0), dried, and mounted on glass slides with Entellan new rapid mounting medium (EMD, Millipore, USA). Microscopic observations were performed using a light microscope (LEICA DM4500 B; Leica microsystems).
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!