Immunostaining of glycolipids on TLC sheets was performed according to the method described by Watarai et al. [23 (link)], with certain modifications. Glycolipids (1 μg) were spotted onto TLC silica gel polyester sheets (100 × 100 mm, Polygram, Macherey-Nagel, Germany) and developed with chloroform/methanol/water/acetic acid (60:40:6:0.2 vol.) at room temperature for 30 min and then air-dried. Glycolipids on the TLC sheets were visualized with a sprayed 5-methylresorcinol (Orcinol, Tokyo Chemical Industry, Tokyo, Japan) solution (2 mg/mL in 2 M H2SO4) and heated at 110°C for 5 min. For immunostaining, TLC sheets were immersed in PBS containing 1% gelatin, 1% polyvinylpyrrolidone (PVP-K30, Nacalai Tesque, Kyoto, Japan), and 1 mM EDTA, followed by immersion in anti-ASGM1 rabbit mAbs (50 ng/mL) or PoAb (1:1,000) solution and allowed to react for 1 h at room temperature. After washing thrice with T-PBS, HRP-conjugated anti-rabbit IgG (1:40,000; The Jackson Laboratory, Bar Harbor, ME, USA) was added. After washing thrice with T-PBS, the TLC sheets were incubated in TMB solution for western blotting (Nacalai Tesque, Kyoto, Japan). Consequently, the bound antibody was visualized in blue.
Pvp k 30
Manufactured by Nacalai Tesque
Sourced in Japan
PVP K-30 is a polyvinylpyrrolidone (PVP) product manufactured by Nacalai Tesque. It is a water-soluble polymer that is commonly used as an excipient in pharmaceutical and cosmetic formulations. The core function of PVP K-30 is to act as a binder, disintegrant, and suspending agent in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Tmb solution for western blotting, by Nacalai Tesque (1 mentions)
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
Ab9051, by Abcam (1 mentions)
Ab9053, by Abcam (1 mentions)
3 protocols using pvp k 30
1
Immunostaining of Glycolipids on TLC Sheets
2
Polymer-based Nanocarrier Formulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PEO (PEO-1NF, molecular weight (MW): 150,000–400,000), HPC (HPC-L, HPC-SL, and HPC-SSL, MW: ~140,000, ~100,000, and ~40,000 respectively), HPMC (HPMC 60SH-50, MW: ~100,000), and PVP (PVP K-30, MW: ~40,000) were used as water-soluble polymers. PEO-1NF was provided by Sumitomo Seika Chemicals Co., Ltd. (Osaka, Japan). HPC-L, HPC-SL, and HPC-SSL were provided by Nippon Soda Co., Ltd. (Tokyo, Japan). PVP K-30 and HPMC were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Sodium lauryl sulfate (SLS) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Purified water was used as the dispersion media. CDTR-PI was provided by Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) (Supplementary Figure S1 ). All other chemicals and solvents were of analytical reagent grade.
3
Immunofluorescence Staining of Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos were fixed in 4% paraformaldehyde in PBS at room temperature (RT) for 20 min after the removal of the zona pellucidae with acid Tyrode’s solution (pH
2.5). After washing three times in PBS containing 0.3% polyvinylpyrrolidone (PVP K-30; Nacalai Tesque), fixed embryos were treated with 0.5% Triton X-100
(Sigma-Aldrich) in PBS at RT for 40 min, and were blocked in PBST containing 1.5% BSA, 0.2% sodium azide, and 0.02% Tween20 (antibody dilution buffer) at RT for
1 h. Embryos were incubated with primary antibody in antibody dilution buffer at 4°C overnight, for H4K20me1 (1:3000 dilution; ab9051, Abcam, Cambridge, UK),
H4K20me3 (1:5000 dilution; ab9053, Abcam), γH2AX (1:200 dilution; AB_315794, Biolegend, San Diego, CA, USA), and FLAG (1:5000 dilution; F1804, Sigma-Aldrich)
staining. Embryos were washed three times in antibody dilution buffer, and then were incubated with appropriate secondary antibody in antibody dilution buffer
(1:500 dilution; Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG, Invitrogen) at RT for 1 h. After stained
with Hoechst 33342 (Sigma-Aldrich), embryos were mounted on slides in 50% glycerol in PBS and signals were observed using a fluorescence microscopy (BX50 or
FSX100, Olympus, Tokyo, Japan). The number of analyzed embryos is given in each figure legend.
2.5). After washing three times in PBS containing 0.3% polyvinylpyrrolidone (PVP K-30; Nacalai Tesque), fixed embryos were treated with 0.5% Triton X-100
(Sigma-Aldrich) in PBS at RT for 40 min, and were blocked in PBST containing 1.5% BSA, 0.2% sodium azide, and 0.02% Tween20 (antibody dilution buffer) at RT for
1 h. Embryos were incubated with primary antibody in antibody dilution buffer at 4°C overnight, for H4K20me1 (1:3000 dilution; ab9051, Abcam, Cambridge, UK),
H4K20me3 (1:5000 dilution; ab9053, Abcam), γH2AX (1:200 dilution; AB_315794, Biolegend, San Diego, CA, USA), and FLAG (1:5000 dilution; F1804, Sigma-Aldrich)
staining. Embryos were washed three times in antibody dilution buffer, and then were incubated with appropriate secondary antibody in antibody dilution buffer
(1:500 dilution; Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG, Invitrogen) at RT for 1 h. After stained
with Hoechst 33342 (Sigma-Aldrich), embryos were mounted on slides in 50% glycerol in PBS and signals were observed using a fluorescence microscopy (BX50 or
FSX100, Olympus, Tokyo, Japan). The number of analyzed embryos is given in each figure legend.
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!