Bioenno Tech, LLC
 
작성일 : 15-07-17
[Bioenno Tech, LLC] Golgi-staining on Free-floating Slices / Selections

 

SliceGolgi/Flxative Kit

 

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Perform Golgi-staining on Free-floating Slices/Selections

 

The sliceGolgi Kit is designed for running Golgi-staining on slices/sections.  The impregnation and

staining can be performed on free-floating sections (50~400 micron thickness). The additional Fixative

Kit uses Bioenno’s aldehyde to process additional samples. The kit has been extensively tested across

many types of brain sections from both rats and mice, and they are:

 

Freshly harvested sections, such as acute slices quickly prepared using tissue chopper.

Organotypic slice cultures, e.g. cultured hippocampal slices and cultured cortical slices.

Artificial cerebrospinal fluid (ACSF)-infused slices that have completed electrophysiological

  recoding and/or drug administration.

Sections derived from brains fixed with Bioenno aldehyde Fixative (Catalog Number: 003780)

  via either perfusion or immersion. The fixed brains can be systematically sliced at 50~400 microns at room

  temperature (RT, 15 to 25 deg C), and the sections can be subjected to Golgi staining alone,

  immunohistochemistry/ immunocytochemistry (ICC) alone, or combined Golgi staining and ICC.

• Additional Fixative Kit allows for processing of more samples. Bioenno suggests purchasing the

  sliceGolgi Kit with the Fixative Kit.

 

The sliceGolgi Kit may be applied to retina, spinal cord, and cultured cells to stain the neuronal

processes.

 

The sliceGolgi Kit yields both reliable and high quality labeling of dendrites and spines (see Figures).  

The impregnation of neurons in 50 to 200 micron thick sections is super-fast, 2-3 days only in some cases.

Generally, the impregnation will take 4-7 days depending on the age and thickness of the sections.  

The kit can be stored in a dark area at 15-25°C for up to 12 months.

 

 

Combined Golgi-staining and immunohitochemistry/immunocytochemistry (ICC)  

Bioenno Tech has successfully combined the use of the sliceGolgi Kit with ICC on the same brain section

(see Figures 4-9). To perform the combined Golgi staining and ICC, fixed sections are first subjected to

Golgi staining, and then to ICC:

 

(1) Tissue Handling: Animals are intracardially perfused with saline followed by a fixative (Catalog

Number: 003780) developed by Bioenno Tech.  Brain are dissected from the skull and post-fixed for 1-3 days

(4°C). Brains are then sectioned at 50~100 micron thickness using a vibratome or similar type of

microtome/microslicer at RT.  Sections are collected in 0.1M PB (pH 7.4) and can be storedin the buffer for

up to one week (4°C).

 

The fixed sections can be used for Golgi staining alone. ICC alone, and combined Golgi staining and ICC.

 

(2) Golgi Staining: Free-floating sections are subjected to Golgi staining using the sliceGolgi Kit at RT (15-

25 °C).  The impregnation of neurons may take 2-5 days.

 

(3) ICC: Free-floating sections can be subjected to standard avidin-biotin complex (ABC) methods.  

The immunoreaction product can be visualized by incubating sections in 3,3′-diaminobenzidine (DAB)

containing H2O2.

 

Notes:The sliceGolgi Kit does not work for frozen tissues. Fixed tissue block can't be frozen

proir to cutting.

 

Product Features

• Suitable for sliced/sectioned tissues (50 to 400 microns thick)

Perform impregnation and staining on free-floating sections

• Novel aldehyde fixtative and enhanced impregnation solution

• Impregnation of neurons is fast (2-7 days)

• Combines both Golgi-staining and immunohistochemistry

• Proven results / user friendly

• Sufficient for up to 1,000 brain sections (50-400 microns thick with dimensions of ~1 x 1.5 cm)

• For in-vitro lab use

• Dedicated technical support

• 12-month warranty

 

 

Proven Results

Proven Results Using the sliceGolgi kit

 

Dendritic branches and spines have been reliably impregnated and stained with the sliceGolgi Kit.

 

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      Fig. 1 - An impregnated and stained neuron in layer II of the frontal cortex

 

The sliceGolgi Kit was used to impregnate and stain the neuron in the frontal cortex of a postnatal day 12

(P12) C57BL mouse. Dendritic spines (arrows) of a neuron in a 200 micron-thick section were well labeled.

Arrowheads indicate filopodia-like protrusions, the immature spines.  The image was taken using a 20x

objective lens.

 

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      Fig. 2 - Dendritic branches of a pyramidal neuron in layer III of the neocortex

 

The sliceGolgi Kit was used to impregnate and stain the oblique dendrites and main branch (stem) of a

pyramidal neuron located in the motor area of the frontoparietal cortex at 20x objective. The C57BL mouse is

12 days old.

 

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                          Fig. 3 - A neuron in the lateral hypothalamus

 

The sliceGolgi Kit was used to impregnate neurons in 100 µm-thick sections under free-floating condition.

The stained neuron with axon terminals was taken (20x objective lens) from the lateral hypothalamic area of

a P12 C57BL mouse. Numerous filopodia-like protrusions (arrowheads) were found on the dendritic

branches.

 

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Fig. 4 

The combined use of sliceGolgi Kit and immunohistochemistry / immunocytochemistry (ICC)

 

The combination of Golgi staining and ICC allows simultaneous visualization of dendritic spines and

immunoreactive products. Generally, free-floating sections were first processed for Golgi-staining, and then

for ICC. For Golgi staining, animals were perfused via the ascending aorta with 0.9% saline solution followed

by freshly prepared fixative (Catalog Number: 003780, Bioenno Tech). Brains were dissected from the skull

and postfixed for 1-3 days (4ºC). Brains were then sectioned at 50-100 μm thickness using a vibratome, and

sections were collected into tissue-culture wells in 0.1 M PB (pH 7.4).

The sliceGolgi Kit (Catalog Number: 003760) was used to impregnate (2-5 days, RT) and stain the

dendrites (black). To perform ICC, free-floating sections were subjected to standard avidin-biotin complex

(ABC) methods. The immunoreaction product was visualized by incubating sections for 8-10 minutes in

0.04% 3,3′-diaminobenzidine (DAB) containing 0.01% H2O2. The image was taken from the hippocampus of

a 2-month old C57 mouse using a 4x objective lens.

 

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        Fig. 5 - Golgi staining and ICC performed on the same brain section

 

Combined Golgi staining and ICC: 1) C57 mouse was intracardially perfused with saline followed by a

Bioenno-developed aldehyde fixative (Catalog Number: 003780). 2) Free-floating sections (50 microns)

were subjected to Golgi staining using the sliceGolgi kit. The time of impregnation was 2 days only at 22

± 1°C. 3) ICC for parvalbumin (PV) was processed using standard avidin-biotin complex methods. The

incubation time of monoclonal mouse anti-PV antibody (1:20,000, MAB1572, Chemicon) was overnight at 22

± 1°C. The image was taken from the thalamus using a 4x objective lens.

 

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      Fig. 6 - Dendritic branches and PV-immunoreactive neurons

 

Golgi staining and ICC were performed on the same brain section. Golgi staining was performed on free-

floating sections (100 µm) using the sliceGolgi Kit. The impregnation time was 3 days at 22 ± 1°C. The

image was taken (63x objective) from the subiculum of a 4-month old C57 mouse.

 

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      Fig. 7 - Golgi-stained dendrites and immuno-labeled neurons

 

The sliceGolgi Kit was used to impregnate and stain the dendritic branches of a cortical neuron in layer III

of the cortex. The immunoreactive neurons were visualized in the same area. The image was taken (63x

objective) from a 4-month old C57 mouse.

 

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Fig. 8 - Golgi-stained neurons and immuno-labeled neurons

 

The sliceGolgi Kit was used to impregnate and stain the dendritic branches of neurons in the deeper layer

of cortex.  The impregnation of neurons in the 100 µm-thick sections took 2 days only at 22 ± 1°C.   

The immunoreactive neurons were visualized in the same area. The image was taken from a 2-month old

C57 mouse using a 20x objective lens.

 

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      Fig. 9 - Golgi-staining and ICC performed on the same brain section

 

The sliceGolgi Kit was used to label dendritic branches. The boxed areas in the top panel, shown at 20x

objective, were magnified (63x) to highlight the dendritic spines (arrows) and immuno-labeled neurons

(arrowheads). These images were taken from the frontoparietal cortex (somatosensory area) of a 2 month

old C57BL mouse.

 


Brochure (PDF)

 

Ordering informations

Catalog No.

Product Name

Size

003760

SliceGolgi

kit*1

003780

Fixative

kit*2

 

*1: up to 1,000 brain sections (50-400 microns thick with dimensions of ~1 x 1.5 cm) / kit

*2: for 2,000 mL